WO2003038082A2

WO2003038082A2 - Novel alkaline protease variants and detergents and cleansers containing these novel alkaline protease variants

Info

Publication number: WO2003038082A2
Application number: PCT/EP2002/011725
Authority: WO
Inventors: Beatrix Kottwitz; Karl-Heinz Maurer; Roland Breves
Original assignee: Henkel Kommanditgesellschaft Auf Aktien
Priority date: 2001-10-31
Filing date: 2002-10-19
Publication date: 2003-05-08
Also published as: HU225362B1; EP1442120A2; HUP0500058A3; US7320887B2; HK1069849A1; HUP0500058A2; JP2005507259A; DE10153792A1; WO2003038082A3; CN1578832A; CN100462434C; US20050003985A1

Abstract

The invention relates to novel alkaline protease variants. These variants have, when enumerating the alkaline protease from Bacillus lentus, variations in amino acid position 61, positions 199 and/or 211 and, optionally, at least one modification that contributes to the stabilization of the molecule, said modification preferably being point mutations in positions 3 and/or 4. Particularly preferred are variants S3T/V4l/G61A/V199l and S3T/V4l/G61A/V199l/L211D of B. lentus alkaline protease. The invention also relates to the possible uses of these enzymes in diverse technical processes and, in particular, to detergents and cleansers containing these novel alkaline protease variants.

Description

New alkaline protease variants and detergents and cleaning agents containing these new alkaline protease variants

The present invention relates to new alkaline protease variants. In the counting of the alkaline protease from Bacillus lentus, these have variations in amino acid position 61, positions 199 and / or 211 and optionally at least one modification which contributes to stabilizing the molecule, preferably point mutations in positions 3 and / or 4. The present invention also relates to the possible uses of these enzymes in various technical processes and in particular washing and cleaning agents with these new alkaline protease variants.

Proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are attributed to the serine proteases due to the catalytically active amino acids. They are naturally formed and secreted by microorganisms, in particular by ßac /// t / s species. They act as non-specific endopeptidases, which means that they hydrolyze any acid amide bonds that are inside peptides or proteins. Their pH optimum is usually in the clearly alkaline range. An overview of this family is provided, for example, by the article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996. Subtilisins are suitable for a variety of technical uses, as components of cosmetics and especially as active ingredients in detergents or cleaning agents.

Proteases are used alongside other enzymes such as amylases, lipases or cellulases as active components in detergents and cleaning agents. They have the ability to break down protein-based soiling on the items to be cleaned, such as textiles or dishes. Due to their higher solubility, the hydrolysis products are washed out with the washing liquor or attacked, dissolved, emulsified or suspended by the other detergent or cleaning agent components. This can result in synergy effects between the enzymes and the other constituents of the detergents and cleaning agents in question. Among the detergent and cleaning agent proteases, subtilisins take due to their favorable enzymatic properties such as stability or pH optimum. The most important of these and the most important strategies for their technical development are listed below.

The basic strategy in the development of detergent proteases is to first isolate enzymes that are naturally formed microbiologically and to test their basic suitability for this application. Optimizations can then be carried out on these molecules. For example, according to application WO 93/07276 A1, the Bacillus spec. Protease 164-A1 available from 164-A1 from Chemgen Corp., Gaithersburg, MD, USA, and Vista Chemical Company, Austin, TX, USA, are suitable for use in detergents and cleaning agents. Other examples are the alkaline protease from Bacillus sp. PD138, NCIMB 40338 from Novozymes (WO 93/18140 A1), which is derived from Bacillus sp. ferm. BP-3376-derived proteinase K-16 from Kao Corp., Tokyo, Japan (US Pat. No. 5,344,770) and according to WO 96/25489 A1 (Procter & Gamble, Cincinatti, OH, USA) the protease from the psychrophilic Organism Flavobacterium balustinum.

Subtilisin BPN ', which comes from Bacillus amyloliquefaciens, or B. subtilis, is from the work of Vasantha ef al. (1984) in J. Bacteriol., Volume 159, pp. 811-819 and by J.A. Wells et al. (1983) in Nucleic Acids Research, Volume _11_, pp. 7911-7925. Subtilisin BPN 'serves, in particular with regard to the numbering of the positions, as a reference enzyme for the subtilisins. For example, the point mutations of application EP 130756 A1 relating to all subtilisins are also given in the numbering of BPN '. Below this is only position 217, which corresponds to position 211 in enzymes according to the invention; no particular exchange is particularly emphasized for this; all are specified, except for the substitution against M, W, C or K; preferably against A or S.

In application CA 2049097 A1 multiple mutants of this molecule are examined, in particular with regard to their stability in detergents and cleaning agents. These include variants with the exchanges Y217K and Y217L and the double mutant S63D / Y217K, that is to say those with exchanges which correspond to positions 211, 61 and 211 of the B. / enfc / s alkaline protease. However, no amino acids were introduced that correspond to one of the proteases of the present application in these positions. Variants obtained by point mutations in the loop regions of this enzyme with reduced binding to the substrate and at the same time increased rate of hydrolysis are presented, for example, in patent applications WO 95/07991 A2 and WO 95/30010 A1. WO 95/07991 A2 relates to the sixth loop of the molecule; Double mutants are disclosed here in which, in addition to a further mutation, the amino acid in position 217 (corresponding to 211 in the beta / etrtus alkaline protease) has mutated to D, for example. Since BPN 'naturally has I in position 205 (corresponding to 199), a maximum of these two positions can be considered here as described above, but always in combination with other mutations in loop regions of the subtilisin and with specific changes in the enzymatic properties. Detergents with such BPN 'variants are disclosed, for example, in patent application WO 95/29979 A1. WO 95/30010 A1 shows further mutations in the other five loop regions, including in position 63 (corresponding to 61), but in this position only for D or E. In contrast, there are two of the amino acid positions considered in the present patent application, namely that Positions 3 and 4, not in loop regions. On the other hand, the numerous exchanges given in these documents do not correlate with stabilizations, in particular not with stabilizing mutations of the subtilisin BPN '.

The protease subtilisin Carlsberg is described in the publications by EL Smith et. al. (1968) in J. Biol. Chem., Volume 243, pp. 2184-2191, and by Jacobs et al. (1985) \ nNucl. Acids Res., Volume _13, pp. 8913-8926. It is naturally formed by Bacillus licheniformis and was, or is or is under the trade name Maxatase ^® from Genencor International Inc., Rochester, New York, USA, and under the trade name Alcalase ^® from Novozymes A / S, Bagsvaerd, Denmark, available. Variants obtainable by point mutations with reduced binding to the substrate and at the same time increased hydrolysis rate are known, for example, from the application WO 96/28566 A2. These are variants in which single or multiple exchanges have been made in the loop regions of the molecule. The only variants tested in washing or cleaning experiments with exchanges in positions which correspond to those of the present application are those of multiple mutants which, among others, exchange G62 (corresponding to position 61 of the β / etrfi / s alkaline protease) to N, D , Q, E, P or S, but not to A, V204 (corresponding to item 199) other amino acids, but not to I and L216 (corresponding to position 211) to 14 other amino acids, including also to D. Thus, through this document only the variations relating to the present application are 3T - because position 3 in subtilisin Carlsberg is naturally occupied by T. - and 211 D described above.

The protease PB92 is naturally derived from the alkaline bacterium Bacillus nov. spec. 92 and Gist-Brocades, Delft, The Netherlands, available under the trade name was Maxacal ^® by the company.. In its original sequence, it is described in patent application EP 283075 A2. Variants of this enzyme obtained by point mutation and which are suitable for use in detergents and cleaning agents are disclosed, for example, in the applications WO 94/02618 A1 and EP 328229 A1. In the first, only exchanges in position 211 for different amino acids, but not for D, are described. The second document discloses that certain areas in which the two residues 61 and 211 are also involved in the substrate binding. However, 61 is not listed among the positions of particular interest for mutagenesis, and an exchange for Y is proposed for 211, which, however, can only increase the washing performance of a corresponding formulation in combination with at least one further exchange.

The subtilisins 147 and 309 are sold under the trade names Esperase ^®, or Savinase ^® by the company. Novozymes. They originally come from β / 7 / us strains which are disclosed in the application GB 1243784 A. Variants of these enzymes further developed by point mutagenesis with regard to their use in detergents and cleaning agents are disclosed, for example, in applications WO 94/02618 A1 (see above), WO 89/06279 A1, WO 95/30011 A2 and WO 99/27082 A1.

The application WO 89/06279 A1 pursued the goal of achieving higher oxidation stability, increased proteolysis rate and improved washing performance. It shows that exchanges in certain positions should change the physical or chemical properties of the subtilisin 147 or 309 molecules (the count of which corresponds to that of the alkaline protease from Bacillus lentus DSM 5483); Among them, item 199 is mentioned, but no special exchange is prescribed. In the application WO 95/30011 A2 variants of subtilisin 309 are presented which point mutations in the loop regions of the molecule exhibit and thus show reduced adsorption on the substrate with a simultaneously increased hydrolysis rate. Positions 61, 199 and 211 also lie in such areas. For position 211, the exchange L211 D is proposed therein, among other things; for positions 61, the exchanges from G to N, D, Q, E, P or S are proposed, for 199 also numerous, but not I. In application WO 99/27082 A1, 309 variants are developed using the example of subtilisin, the Washing performance is improved in that the active loops are increased by inserting at least one amino acid. It is therefore not a question of substitutions as in the present application.

The subtilisin DY was originally developed by Nedkov et al. 1985 in ß / ^' o /. Chem Hoppe-Seyler, Volume 366, pp. 421-430. For example, according to the application WO 96/28557 A2, it can be optimized for use in detergents and cleaning agents via targeted point mutations in the active loops. This gives rise to variants with reduced adsorption and increased hydrolysis rate, including those with exchanges in position 62 (corresponding to 61 in the β / etrtus alkaline protease) from G to N, D, Q, E, P or S, in position 204 (corresponding to 199), but not 204I and numerous in position 216 (corresponding to 211), including D. Since subtilisin DY naturally has T in position 3, a variant 3T / 211 D is hereby described at best.

The enzyme thermitase naturally formed by Thermoactinomyces vulgaris was originally developed by Meloun et al. (FEBS Lett. 1983, pp. 195-200). Variants with reduced absorption and increased hydrolysis rate due to substitutions in the loop regions are described, for example, in the application WO 96/28558 A2. There are exchanges in position 221 (corresponding to 211 in the ß. / Etrfus alkaline protease) for 14 amino acids, including D, and in position 70 (corresponding to 61) from G to N, D, Q, E, P or S described. Since position 209 of the thermitase (corresponding to 199) naturally contains I, at most the variants 1991 and 211 D of the proteases essential for the present application are suggested. In particular, no stabilizations, for example by threonine in position 3 and / or isoleucine in position 4 (according to B. lentus alkaline protease) are suggested. In the corresponding homologous positions 10 and 11, the thermitase has the amino acids S and R (see alignment in WO 91/00345 A1). Thermitase is also a molecule that shows overall significant sequence deviations from the other subtilisins. So the homology between the mature proteins Thermitase and the alkaline protease from ß. lentus DSM 5483 (see below) 45% identity (62% similar amino acids).

Proteinase K is also a protease that forms the alkaline protease from ß. lentus has a comparatively low homology. At the mature protein level, it is only 33% identity (46% similar amino acids). Proteinase K originates from the microorganism Tritirachium album Limber and is from K.-D. Jany and B. Mayer 1985 in / o /. Chem. Hoppe-Seyler, Vol. 366, pp. 485-492. WO 88/07581 A1 discloses the very similar proteases TW3 and TW7, inter alia, for use in detergents and cleaning agents. In application WO 96/28556 A2, numerous exchanges are made for proteinase K, including in position 220 (corresponding to 211 in the β / enft / s alkaline protease) to 14 other amino acids, including D, and in position 68 ( corresponding to 61) from G to N, D, Q, E, P or S. Since in position 208 of proteinase K (corresponding to 199) there is naturally I and in position 4 (corresponding to 3) there is naturally T, thus at most the variations 3T, 1991 and 211 D of the proteases essential for the present application are suggested.

Finally, the Bacillopeptidase F from Bacillus subtilis should also be mentioned, which naturally has the amino acids alanine and isoleucine in positions 61 and 199. Otherwise, it is only slightly similar to the protease variants according to the invention: at the amino acid level, only a homology value of 30% identity or 38% of similar amino acids can be determined. This enzyme is described in the work by Siezen et al. listed, but not yet described or claimed for use in detergents and cleaning agents.

Further proteases suitable for industrial use, in particular in detergents and cleaning agents, are described, for example, in applications EP 199404 A2, EP 251446 A1, WO 91/06637 A1 and WO 95/10591 A1, which are available from Procter & Gamble Comp., Cincinnati, OH, USA, as "Protease A", "Protease B", "Protease C", or "Protease D". The proteases of application EP 199404 are different BPN 'variants which are based on application EP 130756 A1 (see above), but have no variations in the positions relevant to the present application. EP 251446 A1 discloses numerous BPN 'variants, including 217 variants (corresponds to position 211 in the β-lentus alkaline protease); all possible exchanges are named; however, what properties are associated with the 217D variation is not disclosed. According to the application WO 91/06637 A1, "proteases C" are characterized by point mutations of BPN 'in positions 123 and / or 274. The "protease D" is a variant, primarily the protease from Bacillus lentus, which, according to WO 95/10591 A1, have mutations in position 76 (according to BPN 'count, corresponding to position 74 in the β / e / rtus alkaline protease) and additionally other positions, including position 217 (corresponding to 211) However, this does not prescribe an exchange for D. Almost the same applies to US 6017871 A, for example, for use in detergents and cleaning agents and cosmetics and, for example, to US 5677272 A and US 6066611 A for use in bleaching agents: , again in combination with the exchange in position 76, in principle also named the exchange 217D, but is not preferred.

Further known proteases are those under the trade names Durazym ^® , Relase ^® , Everlase ^® , Nafizym, Natalase ^® and Kannase ^® from Novozymes, under the trade names Maxapem ^® , Purafect ^® , Purafect OxP ^® and Properase ^® from Genencor, enzymes available under the trade name Protosol ^® from Advanced Biochemicals Ltd., Thane, India and under the trade name Wuxi ^® from Wuxi Snyder Bioproducts Ltd., China.

One strategy to improve the washing performance of the subtilisins is to introduce point mutations into the known molecules statistically or specifically based on known functions of individual amino acids and to check the variants obtained for their contributions to the washing performance. This strategy is pursued, for example, by patent US 5700676 and application EP 130756 A1 (see above). The only position relating to the present invention describes an exchange in position 217 (corresponding to 211 in the β / enuvs alkaline protease) for all 19 amino acids, either alone or alongside other exchanges which are not relevant for the present application. The same applies to patent US 5801038. In the US 5441882 the method is described to change the enzymatic properties via certain individual exchanges, including alone, or in addition to other exchanges which are not relevant to the present application, however, also in position 217 (corresponding to 211 in the β / enfr / s alkaline protease). Corresponding variants, but in each case only with an exchange, are evident from the patent US 4760025; below this is again only position 217 and without a concrete exchange being disclosed for it.

In order to improve the washing performance of the subtilisins, the strategy of inserting additional amino acids into the active loops was pursued in numerous applications, for example in addition to the aforementioned WO 99/27082 A1, also for the applications which are listed under the numbers WO 00/37599 A1, WO 00/37621 A1 to WO 00/37627 A1 and WO 00/71683 A1 to WO 00/71691 A1 have been published. It should therefore be applicable in principle to all subtilisins that belong to one of the subgroups I-S1 (true subtilisins) or I-S2 (highly alkaline subtilisins).

Another strategy to improve performance is to change the surface charges and / or the isoelectric point of the molecules and thereby their interactions with the substrate. Such variations are presented for example in the patent US 5665587 and the applications EP 405901 A1 and WO 91/00334 A1. Numerous positions are named therein, including 3, 4 and 217 (corresponding to 3, 4 and 211 in the ß. Tenft / s alkaline protease), but without actually revealing corresponding variants. Application WO 91/00345 A1 also refers to these positions in the same sense, likewise without actually specifying corresponding variants. Point mutations for reducing the pH-dependent molecular charge variation are disclosed in WO 92/11348 A1. This may at most affect the exchanges S3T and L211 D which characterize the present application; however, no relevant exchange is specifically disclosed. The application WO 00/24924 A2 derives from this principle a method for identifying variants which should be suitable for use in washing and cleaning agents; all of the variants disclosed have at least one exchange in position 103, preference is given to multiple variants with no exchange relevant to the present application. According to WO 96/34935 A2, the hydrophobicity of the molecules can also be increased for the purpose of improving the performance in washing and cleaning agents, which can have an influence on the stability of the enzyme. From application WO 99/20727 A2, subtilisin variants emerge as can be obtained by a method of application WO 00/24924 A2: they all contain at least one exchange in position 103, combined with a large number of other possible exchanges, but none of them in the position corresponding to position 61 of the protease from ß. lentus corresponds. Multiple variants with at least six exchanges are preferred, including positions 205 and 217 (corresponding to 199 and 211 in the beta / erectus alkaline protease); specifically, only two of more than 50 of these variants have the exchange 1991 relevant for the present application. The same mutants are disclosed in applications WO 99/20723 A2 and WO 99/20726 A2 for detergents and cleaning agents which additionally contain an amylase or bleach contain.

A modern direction of enzyme development is to combine elements from known, related proteins by statistical methods to new enzymes that have previously unattained properties. Such processes are also summarized under the generic term Directed Evolution. These include, for example, the following methods: the StEP method (Zhao et al. (1998), Nat. Biotechnol., Volume 16, pp. 258-261), random priming recombination (Shao et al., (1998), Nucleic Acids Res ., Volume 26, pp. 681-683), DNA shuffling (Stemmer, WPC (1994), Nature, volume 370, pp. 389-391) or RACHITT (Coco, WM et al. (2001), Nat. Biotechnol , Volume 19, pp. 354-359).

Another, in particular complementary, strategy is to increase the stability of the proteases in question and thus to increase their effectiveness. Stabilization via coupling to a polymer has been described for proteases which are used in cosmetics, for example in US Pat. No. 5,230,891; it goes hand in hand with better skin tolerance. In contrast, stabilization by point mutations is more common, especially for detergents and cleaning agents. According to the patents US 6087315 and US 6110884 proteases can be stabilized by exchanging certain tyrosine residues for others. WO 89/09819 A1 and WO 89/09830 A1 describe thermostable BPN 'variants which have the substitutions for K or L in positions 217 (corresponding to 211 in the β. Fenfcts alkaline protease) and in addition to 217K in Position 63 (corresponding to position 61) the exchange S63D. Other described options for stabilization via point mutagenesis are, for example:

- Exchange of certain amino acid residues for proline according to WO 92/19729 A1, or EP 583339 B1 and US patent 5858757 and according to EP 516200 A1;

- Introduction of more polar or more charged groups on the surface of the molecule according to EP 525610 A1, EP 995801 A1 and US patent 5453372, inter alia in the position that the V4 of the protease from ß. lentus corresponds; on the other hand, with the change V4I, as in the present application, a less polar amino acid is introduced;

- Strengthening the binding of metal ions, in particular via mutagenesis of the calcium binding sites, for example according to the teaching of the applications WO 88/08028 A1 and WO 88/08033 A1;

- Blockage of the autolysis by modification or mutagenesis, for example according to the patent US 5543302.

A combination of several stabilization strategies is disclosed in the application EP 398539 A1. Accordingly, subtilisins can be stabilized and their contribution to the washing or cleaning performance improved by (1.) replacing amino acids of the calcium binding sites with more negative ones, (2.) deleting or mutating natural consequences of Asn-Gly, (3. ) Met residues are exchanged for others and (4.) certain amino acids are also substituted that are close to the catalytic center. None of the first three options apply to the variants of the present application according to the invention. Items 61 and 211 are affected by the fourth option. However, it is proposed here to replace the amino acids naturally occurring in these positions (S63, or Y217 in the case of subtilisin BPN ') by G or L, respectively. In contrast, in the molecules of the present application it is precisely these positions which are occupied by amino acids other than G or L.

Further possibilities of stabilizing subtilisins, in particular those derived from that of Bacillus lentus, via point mutagenesis are specified in the patents US 5340735, US 5500364, US 5985639 and US 6136553. The mutated positions are determined by analyzing the three-dimensional structure. Variants in items 61 and 211 are not described in any of these documents. For example, documents EP 755999 A1 and WO 98/30669 A1 show that proteases, in particular performance-enhanced proteases, can be used in detergents and cleaning agents to improve the washing or cleaning performance together with α-amylases and other detergent enzymes. For example, it is known from application WO 97/07770 A1 that some of those which have previously been established as detergent proteases (see below) are also suitable for cosmetic purposes. Another possible use of proteases is presented, for example, in the application EP 380362 A1. This relates to organic chemical syntheses, for which, according to this application, such subtilisins are said to be suitable which, via point mutagenesis after the .beta. / eπfus-alkaline protease count alone or alongside other mutations in positions 61 (by mutation to D) and / or 211 (by mutation to K or L) are stabilized. In this context, too, no exchange relevant to the present invention has been described.

For the alkaline proteases from ß. lentus are highly alkaline proteases from Bacillus species. According to the application WO 91/02792 A1, one of these strains has been deposited under the number DSM 5483; the sequences and biochemical properties of the wild-type enzyme are also disclosed therein. Variants of this enzyme which are to be obtained by point mutation and are suitable for use in detergents and cleaning agents are disclosed in WO 92/21760 A1 and WO 95/23221 A1.

The wild-type enzyme comes from a producer that was originally obtained by screening for alkaliphilic Bacillus strains and shows a relatively high stability against oxidation and the action of detergents. The applications WO 91/02792 A1, or the patents EP 493398 B1 and US 5352604, describe its heterologous expression in the host Bacillus licheniformis ATCC 53926. In the claims of said U.S. patent, positions 208, 210, 212, 213 and 268 are characteristic of this. called lentus- alkaline protease; these correspond to positions 97, 99, 101, 102 and 157 in the count of the mature protein, in which this enzyme differs from the mature Savinase ^® . The three-dimensional structure of this enzyme is described in the publication Goddette et al. (1992) J. Mol. Bioi, Volume 228, pp. 580-595: "The crystal structure of the Bacillus lentus alkaline protease, Subtilisin BL, at 1.4 A resolution ".

Application WO 92/21760 A1, or patent US 5340735, also discloses the wild-type enzyme β. / enfc / s-Alkaline protease (formed by ß. lentus DSM 5483) under SEQ ID NO: 52 in its amino acid sequence and under SEQ ID NO: 106 in its nucleotide sequence. In addition, 51 different variants derived from this are disclosed in this application, which differ from the wild type in one or more positions and have thereby been stabilized. These include the S3T, V4I and V199I exchanges. According to this application, the variant M131 with the exchanges S3T / V4I /, deposited under the designation ATCC 68614 at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (http://www.atcc.org). A188PΛ 193MΛ 199I most preferred. This serves as the starting enzyme for the present application (see Example 1) and is also included in the sequence listing under SEQ ID NO. 1 and 2 specified in their DNA or amino acid sequence. They are all derived from the alkaline protease from Bacillus lentus DSM 5483. By point mutations at other positions, stability-improved variants emerge, for example, from the US Patents US 5500364 and US 5985639 derived from the WO document.

From application WO 95/23221 A1, for improved use ß for use in detergents and cleaning agents by targeted point mutagenesis. lentus alkaline protease variants, which are to be regarded as further developments of the aforementioned molecules. Some of them also have the three exchanges S3T, V4I and V199I. In addition, they all have two or three other point mutations compared to the wild-type enzyme from ß. lentus DSM 5483. Sometimes they carry an additional mutation in position 211, namely 211D (variants F49, F54 and F55). Consequently, this application, or the associated US patents US 5691295, US 5801039 and 5855625, claim variants with the exchanges 211 D and 211E. The associated strategy, namely to specifically change the charge conditions near the substrate binding pocket, is illustrated by the patent US 6197589. As all of this work, which has been carried out over a long period of time, shows, there is a great need for proteases that can be used technically, some of which differ drastically, some only in a few positions from previously known proteases. They cover a wide range from very drastic to very subtle differences in performance. This is particularly evident in their use in detergents and cleaning agents. When developing them, the possibly calculable enzymatic properties cannot readily be used to infer the behavior of these enzymes, for example in the context of a detergent or cleaning agent formulation. Other factors such as stability against high temperatures, oxidizing agents, denaturation by surfactants, folding effects or desired synergies with other ingredients play a role here, which can often only be developed experimentally.

The object underlying the present invention was to find subtilisins which perform better in technical applications. In particular, those should be found that improve the washing or cleaning performance of detergents and / or cleaning agents. One part of the task was not only to improve the proteases with regard to their hydrolysis activity, but also to keep them stable in the corresponding formulations.

Further subtasks consisted of providing nucleic acids which code for such proteases and vectors, host cells and production processes which can be used to obtain such proteases. Corresponding agents, in particular washing and cleaning agents, corresponding washing and cleaning processes and corresponding uses for such proteases should also be made available. Finally, technical applications for the proteases found should be defined.

Surprisingly, it was found that an exchange of the amino acid glycine in position 61 for another amino acid, in particular for an aliphatic amino acid and very particularly for alanine, makes an increased contribution to the washing performance. The contributions of other exchanges, such as that for isoleucine in position 199 and / or other defined amino acid residues such as aspartic acid in position 211, presumably benefit from this through an enzymatic effect. This increased effectiveness is enhanced by the amino acids threonine and isoleucine in positions 3 and 4, presumably via a stability effect.

According to the invention, this object is therefore achieved by alkaline proteases of the subtilisin type, which are characterized in that, according to the counting of the subtilisin from Bacillus lentus in position 61, an exchange for one of the amino acids alanine, valine, leucine, isoleucine, cysteine relative to the starting enzyme , Methionine, phenylalanine, tyrosine, tryptophan, threonine, histidine, lysine or arginine, preferably against one of the amino acids alanine, valine, leucine or isoleucine, particularly preferably against alanine.

Solutions in which an isoleucine is present in position 199 in addition to the exchange in position 61 are increasingly preferred; those in which one of the amino acids identified above in position 61, isoleucine in position 199, aspartic acid in position 211 and threonine and / or isoleucine in position 4 for stabilization. Particularly preferred solutions are such proteases, which differ from the β. Derive / eπfus alkaline protease, in particular one of the two variants ß. / errfi / s-Alkaline protease S3T / V4I / G61A / V199I or ß. / enft / s-Alkaline protease S3TΛ 4I / G61A / V199I / L211 D. This object of the invention also includes further developments and derivatives of the proteases mentioned.

The present invention provides nucleic acids which code for proteases according to the invention, as well as vectors, host cells and production processes which can be used to obtain such proteases as solutions to the subtasks and thus as separate objects of the invention. Corresponding agents, in particular detergents and cleaning agents, corresponding washing and cleaning processes and corresponding uses for such proteases are also provided. Finally, technical uses for the proteases found are defined.

For the purposes of the present application, a protein is to be understood as a polymer which is composed of the natural amino acids and has a largely linear structure and usually assumes a three-dimensional structure to perform its function. In the present application, the 19 proteinogenic, naturally occurring L- Amino acids labeled with the internationally used 1- and 3-letter codes.

The combination of one of these names with a number indicates for each protein which amino acid residue it carries in the respective position. For example, S3 stands for a serine residue in position 3, starting with the count at the N-terminus of the protein in question. A point mutation at this point, for example against the amino acid threonine, is abbreviated according to this nomenclature with the designation S3T. To indicate variants with multiple point mutations, these exchanges are separated from one another with slashes. The variant S3TΛ / 4I is therefore characterized in that the serine previously in position 3 has been replaced by a threonine and the valine in position 4 by an isoleucine.

Unless otherwise stated, the position specifications of the present invention relate to the respective mature forms of the proteins in question, that is to say without the signal peptides (see below).

For the purposes of the present application, an enzyme is to be understood as a protein which has a specific biochemical function. For example, proteolytic enzymes or enzymes with a proteolytic function are generally to be understood as those which hydrolyze the acid amide bonds of proteins, in particular those which are located inside the proteins and which can therefore also be referred to as endo-peptidases. Subtilisin proteases are endo-peptidases that are naturally formed by gram-positive bacteria and mostly secreted, or are derived from them, for example using molecular biological methods, and can be homologized with the natural subtilisin proteases via sub-areas such as structure-forming or function-bearing regions , They are described, for example, in the article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996.

Numerous proteins are formed as so-called pre-proteins, i.e. together with a signal peptide. This is to be understood as the N-terminal part of the protein, the function of which mostly consists in the removal of the protein formed to ensure the producing cell in the periplasm or the surrounding medium and / or its correct folding. The signal peptide is then cleaved from the rest of the protein under natural conditions by means of a signal peptidase, so that this exerts its actual catalytic activity without the N-terminal amino acids initially present. According to FIG. 1 in WO 91/02792 A1, the preprotein of Bacillus lentus DSM 5483 subtilisin contains 380 amino acids. The mature protein, however, only 269; the count begins with the first amino acid of the mature protein, in this case with the alanine, which would have the number 112 according to the sequence of the preprotein. According to SEQ ID NO. 1 of the present application is the signal peptide of the subtilisin from β. licheniformis ATCC 68614 111 amino acids long and the mature peptide 269 amino acids. Without this subdivision, the complete protein is 380 amino acids in length, as from SEQ ID NO. 2 emerges. The same applies to the particularly preferred embodiments according to the sequence listing.

Due to their enzymatic activity, the mature peptides, that is to say the enzymes processed after their production, are preferred over the pre-proteins for technical applications.

Pro-proteins are inactive precursors of proteins. Their precursors with a signal sequence are called pre-pro proteins.

For the purposes of the present application, nucleic acids are understood to mean the molecules which are naturally built up from nucleotides and serve as information carriers and which code for the linear amino acid sequence in proteins or enzymes. They can be present as a single strand, as a single strand complementary to this single strand or as a double strand. As the naturally more permanent information carrier, the nucleic acid DNA is preferred for molecular biological work. In contrast, an RNA is formed for the implementation of the invention in a natural environment, such as, for example, in an expressing cell, which is why RNA molecules essential to the invention also represent embodiments of the present invention.

The information unit of a nucleic acid corresponding to a protein is also referred to as a gene in the sense of the present application. In DNA, the sequences of both complementary strands are closed in all three possible reading frames consider. It should also be taken into account that different codon triplets can code for the same amino acids, so that a certain amino acid sequence can be derived from several different and possibly only slightly identical nucleotide sequences (degeneracy of the genetic code). In addition, different organisms have differences in the use of these codons. For these reasons, both amino acid sequences and nucleotide sequences have to be included in the consideration of the protected area, and specified nucleotide sequences are only to be regarded as exemplary coding for a specific amino acid sequence.

It is possible for a person skilled in the art to use known methods today, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and / or protein chemical methods, to produce complete genes using known DNA and / or amino acid sequences. Such methods are known, for example, from the "Lexicon of Biochemistry", Spectrum Academic Publishing House, Berlin, 1999, Volume 1, pp. 267-271 and Volume 2, pp. 227-229. This is possible in particular if one of the The genes in question can be easily synthesized, cloned and, if desired, further processed from such strains using PCR primers which have been synthesized on the basis of a known sequence, such as, for example, site-directed or randomly controlled mutagenesis.

Changes in the nucleotide sequence, such as can be brought about, for example, by known molecular biological methods, are referred to as mutations. Depending on the type of change, deletion, insertion or substitution mutations are known, for example, or those in which different genes or parts of genes are fused to one another (shuffling); these are gene mutations. The associated organisms are called mutants. The proteins derived from mutant nucleic acids are called variants. For example, deletion, insertion or substitution mutations or fusions lead to deletion, insertion or substitution mutations or fusion genes and at the protein level to corresponding deletion, insertion or substitution variants or fusion proteins. For the purposes of the present invention, vectors are understood to mean elements consisting of nucleic acids which contain a gene of interest as the characteristic nucleic acid region. They are able to establish this in a species or a cell line over several generations or cell divisions as a stable genetic element that replicates independently of the rest of the genome. Especially when used in bacteria, vectors are special plasmids, i.e. circular genetic elements. In genetic engineering, a distinction is made on the one hand between such vectors, which are used for storage and thus to a certain extent also for genetic engineering work, the so-called cloning vectors, and on the other hand, those which fulfill the function of realizing the gene of interest in the host cell, i.e. the expression of the enable relevant protein. These vectors are called expression vectors.

By homologation, that is to say comparison with known enzymes, as is carried out, for example, via an alignment, the enzymatic activity of an enzyme under consideration can be deduced from the amino acid or nucleotide sequence. This can be qualitatively or quantitatively modified by other areas of the protein that are not involved in the actual reaction. This could affect enzyme stability, activity, reaction conditions or substrate specificity, for example.

The term a proteolytic enzyme or that of a protease is therefore to be understood, in addition to the functions of the few amino acid residues of the catalytically active center, to all functions which actually affect the action of the entire remaining protein or part or more parts of the remaining protein result in catalytically active areas. Even such modifying functions or partial activities, provided they support a proteolysis reaction, are regarded as proteolytic activity in the sense of the invention. Such auxiliary functions or partial activities include, for example, the binding of a substrate, an intermediate or end product, the activation or the inhibition or mediation of a regulating influence on the hydrolytic activity. For example, this can also be the formation of a structural element that is remote from the active center. The second prerequisite for the fact that it is a proteolytic protein according to the invention is, however, that the chemical behavior of the actually active residues alone or additionally by the Exposure of the modifying parts results in hydrolysis of peptide bonds. It is also possible for the activities of other proteases to be qualitatively or quantitatively modified by one or more parts, for example the protein according to the invention. This influence on other factors is also considered to be proteolytic activity. Proteolytically active enzymes are also those whose activity is blocked at a given point in time, for example by an inhibitor. Their fundamental suitability for the corresponding proteolysis reaction is crucial.

Fragments are understood to mean all proteins or peptides that are smaller than natural proteins or those that correspond to fully translated genes and that can also be obtained synthetically, for example. Based on their amino acid sequences, they can be assigned to the complete proteins concerned. For example, they can take on the same structures or have proteolytic or sub-activities, such as complexing a substrate. Fragments and deletion variants of parent proteins are basically the same; whereas fragments represent smaller fragments, the deletion mutants tend to lack only short areas, and thus only partial functions.

For the purposes of the present application, chimeras or hybrid proteins are understood to mean those proteins which are composed of elements which naturally originate from different polypeptide chains from the same organism or from different organisms. This procedure is also called shuffling or fusion mutagenesis. The purpose of such a fusion can be, for example, to bring about or to modify an enzymatic function with the aid of the protein part according to the invention fused into it. In the context of the present invention, it is immaterial whether such a chimeric protein consists of a single polypeptide chain or several subunits, over which different functions can be distributed. To implement the latter alternative, it is possible, for example, to split a single chimeric polypeptide chain into several post-translationally or only after a purification step by means of a targeted proteolytic cleavage.

Proteins obtained by insertion mutation are to be understood as those variants which have been obtained by methods known per se by inserting a nucleic acid or protein fragment into the starting sequences. she are assigned to the chimeric proteins because of their principle similarity. They differ from those only in the size ratio of the unchanged protein part to the size of the entire protein. The proportion of foreign protein in such insertion-mutated proteins is lower than in chimeric proteins.

Inversion mutagenesis, i.e. a partial reversal of the sequence, can be viewed as a special form of both deletion and insertion. The same applies to a regrouping of different parts of the molecule that deviates from the original amino acid sequence. It can be viewed both as a deletion variant, as an insertion variant, and as a shuffling variant of the original protein.

In the context of the present application, derivatives are understood to mean those proteins whose pure amino acid chain has been chemically modified. Such derivatizations can take place, for example, biologically in connection with protein biosynthesis by the host organism. Technically, molecular biological methods can be used for this. However, they can also be carried out chemically, for example by chemically converting a side chain of an amino acid or by covalently binding another compound to the protein. Such a compound can also be, for example, other proteins which are bound to proteins according to the invention, for example, via bifunctional chemical compounds. Such modifications can, for example, influence the substrate specificity or the binding strength to the substrate or can temporarily block the enzymatic activity if the coupled substance is an inhibitor. This can be useful for the storage period, for example. Derivatization should also be understood to mean covalent binding to a macromolecular carrier.

For the purposes of the present invention, all enzymes, proteins, fragments and derivatives, unless they need to be explicitly addressed as such, are summarized under the generic term proteins.

The performance of an enzyme is understood to mean its effectiveness in the technical area under consideration. This is based on the actual enzymatic activity, but also depends on other factors relevant to the respective process. This includes, for example, stability, substrate binding, interaction with the material carrying the substrate or interactions with other ingredients, especially synergies.

For the purposes of the present application, the washing performance or the cleaning performance of an agent is to be understood as the effect that the agent in question has on the soiled articles, for example textiles or objects with hard surfaces. Individual components of such agents, for example individual enzymes, are assessed with regard to their contribution to the washing or cleaning performance of the entire agent. Because the enzymatic properties of an enzyme cannot be used to infer its contribution to the washing performance of an agent. Stability, substrate binding, binding to the items to be cleaned or interactions with other ingredients of the detergents, in particular synergies in the removal of the contaminants, play a role here as further factors.

With regard to the task at hand, the present patent application pursued the strategy of subtilisin from Bacillus lentus DSM 5483, in particular in relation to the molecules disclosed in applications WO 91/02792 A1, WO 92/21760 A1 and WO 95/23221 A1, and very particularly in relation to the Variants M131 S3T / V4I / A188PΛ / 193M / V199I and F49 S3TΛ 4I / A188P / V193MΛ / 199I / L211 D for use in detergents and cleaning agents. The associated technical teaching can accordingly also be applied to other, in particular highly related proteases, very particularly of the subtilisin type.

Positions 3, 4, 61, 199 and 211 of the mature proteins according to the counting of the subtilisin from Bacillus lentus DSM 5483 (WO 92/21760 A1) are particularly important to the invention. According to Table 1, these can be homologized with those from the most important subtilisins; this homologation can be applied to all other subtilisins. For example, in the article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996, an alignment of over 20 subtilisins relative to the known sequence of subtilisin BPN '. Table 1: Homologation of the five positions that are particularly important to the invention.

An alignment of the amino acid sequences of the β according to the invention. / enfr / s-Alkaline protease variants S3T / V4I / G61A / V199I and S3TΛ / 4I / G61AΛ / 199I / L211 D with these most important subtilisins described in the introduction, namely Subtilisin 309 (Savinase ^® ), Subtilisin PB92, Subtilisin Carlsberg and Subtilisin BPN 'is also shown in Figure 1 of this patent application.

The transferability of the teaching of the present invention is based on the high structural similarities between the subtilisins and the largely identical reaction mechanism. It can therefore be expected that the point mutations mentioned will each have comparable effects in the context of the molecule in question. In particular, on the basis of the teaching of the present patent application, it is to be expected that such subtilisins, which have already been developed in the prior art with regard to their use in detergents and cleaning agents, will continue by adopting these point mutations with regard to their contributions to the washing or cleaning services can be improved.

For position 61 as counted this. / etrtus alkaline proteases have been described in the prior art (see above) for exchanges for the amino acids N, D, Q, E, P and S, in particular in combination with other point mutations not relating to the present invention. Substitutions against the neutral amino acid threonine containing a hydroxyl group, against the basic amino acids histidine, lysine, arginine, against the aromatic amino acids phenylalanine, tyrosine, Tryptophan, against the sulfur-containing amino acids cysteine, methionine and against the aliphatic amino acids alanine, valine, leucine, isoleucine have not yet been described; in particular not with the aim of thereby improving the performance of the enzyme, specifically its contribution to the washing or cleaning performance of a corresponding agent. This is achieved by the present patent application.

According to the present invention, the performance-improving exchange in position 61 is preferably one for an aliphatic amino acid, namely alanine, valine, leucine or isoleucine, particularly preferably for alanine. Because this characterizes the variants examined in the examples. / enfrts-Alkaline protease S3TΛ / 4I / G61A / V199I and ß. teπft / s-alkaline protease

S3T / V4I / G61A / V199I / L211 D. Surprisingly, it was found that an exchange of the amino acid glycine in position 61 for another amino acid, in particular for an aliphatic amino acid and very particularly for alanine, makes an increased contribution to washing performance.

As the variant of a subtilisin from Bacillus lentus, which to the variant ß. / etrtws-Alkaline Protease S3TΛ / 4I / G61AΛ 199I / L211 D has the highest degree of agreement, the ß known from WO 95/23221 A1. / etrfi / s-Alkaline protease variant F49 with the characteristic exchanges

S3TΛ 4I / A188PΛ / 193M / V199I / L211 D can be viewed. The variant closest to S3T / V4I / G61AΛ / 199I is accordingly the β known from WO 92/21760 A1 and referred to in WO 95/23221 A1 as M131. / entos alkaline protease variant S3TΛ / 4I / A188P / V193MΛ 199I.

Instead of the two exchanges A188P and V193M, a variation of position 61 has thus taken place in the present invention. As can be seen, for example, from application WO 95/30011 A2, amino acid 193 is located in beta. lentus subtilisins at the beginning of the 6th loop, while amino acid 188 is not a loop but the compact region of the protein in between. In this respect, both mutations lie in structurally different areas of the molecule. It was surprisingly found with the present invention that a reversion of the two positions 188 and 193 to the amino acids of the wild type and an additional mutation in the position 61, that is to say in the 1st loop, gives an enzyme which previously known enzymes, especially the previously known variants of ß. / enfx / s-Alkaline protease is superior in its washing or cleaning performance. This is demonstrated in particular by the results shown in Examples 3, 5 and 7.

For example, in applications CA 2049097 A1, EP380362 A1 and WO95 / 30010 A1 (see above) variations in position 61 are proposed, but only for acidic amino acids. In a series of applications for point mutagenesis on subtilisins, including WO 96/28556 A2 and in particular WO 95/30011 A2 (see above), such exchanges are also described for position 61 (naturally glycine in most subtilisins) (N, D, Q , E, P and S), which mean very drastic changes for the molecule locally and probably also influence the substrate interactions exerted by the first loop. In contrast, EP 398539 B1 even proposes a mutagenesis which leads to the fact that the amino acid glycine is only introduced at this point in the subtilisin in question, whereas it is naturally present there in the subtilisin from Bacillus lentus. In view of this prior art, it is surprising that a change in this position, in particular to an amino acid with an aliphatic side chain, and very particularly to alanine, has an advantageous effect on the reaction carried out by the enzyme.

From the point of view of technical application, in particular in detergents and cleaning agents, the surprising thing is that this leads to an improvement in performance, in particular to an improvement in the contribution of such enzymes to the washing and cleaning performance of various soils. This is shown in the exemplary embodiments 2 to 7 of the present application.

According to these observations, those variants are preferred which, in addition to the exchanges mentioned in position 61, have the amino acid isoleucine according to the counting of the subtilisin from Bacillus lentus in position 199. The influence of this exchange on the enzymatic properties of the subtilisin from Bacillus lentus DSM 5483 have been described, for example, in WO 92/21760 A1. Further preferred are alkaline proteases of the subtilisin type, which are characterized in that according to the counting of the subtilisin from Bacillus lentus in position 199 isoleucine, in position 211 aspartic acid and in position 61 one of the amino acids alanine, valine, leucine, isoleucine, cysteine , Methionine, phenylalanine, tyrosine, tryptophan, threonine, histidine, lysine or arginine, preferably alanine, valine, leucine or isoleucine, particularly preferably alanine. This applies to both natural and mutagenesis-derived molecules with these amino acids in these positions.

The contribution of the amino acid residue aspartic acid in position 211 to increasing the washing or cleaning performance of a corresponding agent can be seen, for example, from example 10 of application WO 95/23221 A1.

Alkaline proteases according to the invention are preferably characterized in that they have at least one stabilization. Because this increases their stability during storage and / or during their use, so that the advantageous effect of the aforementioned amino acids or amino acid exchanges lasts longer and is thus enhanced.

The stability of proteases according to the invention can be increased, for example, by coupling to polymers. Such a method is described for example in the patent US 5230891. It requires that the proteins are linked to such polymers via a chemical coupling step before they are used in appropriate agents.

Stabilizations that are possible via point mutagenesis of the molecule itself are preferred. Because these do not require any further work steps after the protein extraction. Some point mutations suitable for this are known per se from the prior art. According to the patents US 6087315 and US 6110884 proteases can be stabilized by exchanging certain tyrosine residues for others. Transferred to proteins according to the invention derived from Bacillus lentus, this would replace the tyrosine residues in positions 89, 161, 165, 208 and 257 according to SEQ ID NO. 2 mean; the other two positions specified there are in ß. / enfc / s-Alkaline protease already ingested by tyrosine. Other options include:

- The exchange of certain amino acid residues for proline according to EP 583339 B1; that would be for enzymes that are different from ß. derive lentus, which mean exchanges S55P, A96P, A166P, A188P and / or S253P);

- The introduction of more polar or charged groups on the surface of the molecule according to EP 995801 A1;

- Change in the binding of metal ions, in particular the calcium binding sites, for example according to the teaching of the applications WO 88/08028 A1 and WO 88/08033 A negatively charged amino acids are exchanged. After the publication by D.W. Goddette et al. (1992) in J. Mol. Bioi, volume 228, pages 580-595, the subtilisin from Bacillus lentus has the following two calcium binding sites: Ca1 (with high binding affinity), comprising the positions 2 Q (s), D40 (s, 2x), L73 *, N75 (m), I77 (s), V79 (m) and the site Ca3 (with low binding affinity) comprising positions A168, A163, Y165, water 273, 317; each according to the numbering of the Bacillus lentus subtilisin.

- According to the teaching of the application WO 88/08033 A1, point mutations would have to be introduced simultaneously in at least one of the sequences of the two residues arginine / glycine for stabilization via the calcium bond; in subtilisins from Bacillus lentus, for example, this affects the NG sequences in positions 60/61, 115/116 and 212/213.

- According to the patent US 5453372, proteins can be protected against the influence of denaturing agents such as surfactants by certain mutations on the surface; correspond to the positions given there in ß. / en us-Alkaline protease positions 134, 155, 158, 164, 188 and / or 189.

Further comparable possibilities are given in the patents US 5340735, US 5500364, US 5985639 and US 6136553.

Such a stabilization for alkaline proteases according to the invention is preferably that by the amino acid threonine in position 3 according to the counting of the subtilisin from Bacillus lentus. This is because, for example, Table 3 of application WO 92/21760 A1 shows that this exchange stabilizes the molecule with respect to the wild-type enzyme both against elevated temperature and against the action of surfactants. The N-terminus of the molecule, to which positions 3 and 4 belong, lies on the surface after processing, namely on End of the column with the active center. This loose end is in contact with the rest of the molecule in particular via non-covalent interactions and thus contributes to the maintenance of the globular structure. Without being bound by this theory, one can assume that any mutations that limit the flexibility of this loose end contribute to the stability of the entire molecule.

Such a stabilization for alkaline proteases according to the invention is likewise preferably those by the amino acid isoleucine in position 4 according to the counting of the subtilisin from Bacillus lentus. The stabilizing effect of this exchange is also shown in Table 3 of application WO 92/21760 A1.

The molecule is particularly preferably stabilized via the exchange for threonine in position 3 and that for isoleucine in position 4, or its performance profile is modified.

For corresponding thereto subtilisins according to the invention showed in the examples of the present application in corresponding washing or cleaning agent formulations performance improvements over the process known from the prior art enzyme Savinase ^®, which does not have this stabilization. Without being bound by this theory, one could assume that the stability of the variants in question helps to keep the enzymes active in the wash liquor sufficiently long and thus supports the improved performance.

In addition, each of these exchanges can also improve the performance of the molecule in a different way, in particular in detergents and cleaning agents, for example by interacting with a substrate or with other agents corresponding to the substance.

Particularly preferred are the embodiments in which the alkaline protease according to the invention is characterized in that, according to the counting of the Bacillus lentus subtilisin, it has threonine in position 3, isoleucine in position 4, alanine in position 61 and isoleucine in position 199. The contribution of such a variant to the washing or cleaning performance of a corresponding agent is documented in Examples 3, 5 and 7 of the present application.

The embodiments are particularly preferred in which the alkaline protease according to the invention is characterized in that according to the counting of the subtilisin from Bacillus lentus in position 3 threonine, in position 4 isoleucine, in position 61 alanine, in position 199 isoleucine and in position 211 Has aspartic acid.

The contribution of such a variant to the washing or cleaning performance of a corresponding agent is documented in Examples 2, 4 and 6 of the present application.

Such variants are preferably derived from a Bacillus subtilisin, in particular from Bacillus / enfc / s subtilisin.

Because Bacillus proteases have favorable properties from the outset for various technical applications. This includes a certain stability against elevated temperature, oxidizing or denaturing agents. In addition, microbial proteases have the greatest experience with regard to their biotechnological production, for example in the construction of cheap cloning vectors, the selection of host cells and fermentation conditions or the assessment of risks such as allergenicity.

Especially the subtilisins from Bacillus lentus, or the subtilisins, which are derived from its naturally formed proteases, have been established in the prior art, for example for use in detergents and cleaning agents. These include the proteases mentioned at the beginning, Subtilisin 147, Subtilisin 309 and ß. / entas alkaline protease. The wealth of experience that has been acquired for the production and use of these proteases benefits further developments of these enzymes according to the invention. This includes, for example, their compatibility with other chemical compounds, such as the ingredients of detergents or cleaning agents. A suitable starting strain for this can be found under the deposit number DSM 5483 at the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, 38124 Braunschweig (http://www.dsmz.de) and for example in the applications WO 91/02792 A1, WO 92/21760 A1 or WO 95/23221 A1 described ß. lentus strain can be used. Such variants of this or of related strains can be produced using standard molecular biological methods, such as, for example, PCR and point mutagenesis methods known per se.

The particularly preferred variants described in the examples are of the ß via the procedure shown in example 1. / e / 7uvs-Alkaline protease of ß. lentus, which has been deposited under the designation ATCC 68614 with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (http://www.atcc.org). This enzyme is in the sequence listing of the present application under SEQ ID NO. 1, and 2 disclosed in its nucleotide sequence and its amino acid sequence.

This sequence can be used, for example, for the construction of primers in order to produce a nucleic acid coding for such a protease from DNA preparations of gram-positive bacteria, preferably gram-positive bacteria such as Bacillus lentus, if necessary, for example by methods known per se, for example to mutagenize and express using the teaching of the present application. Due to the degeneracy of the genetic code, numerous other nucleic acids are also conceivable, which also code for this variant and are equally preferred alternatives within this subject matter of the invention.

These include in particular the ß. / enfrys-Alkaline protease S3T / V4I / G61A / V199I according to the sequence listing under SEQ ID NO. 3 indicated nucleotide sequence, but especially according to the SEQ ID NO. 4 given amino acid sequence. Because their improved contribution to the washing performance is documented with the corresponding application examples of the present application.

Also preferred according to the above is an alkaline protease which is characterized in that it is one of the subtilisin from Bacillus lentus DSM 5483, or ATCC 68614 derived subtilisin, especially ß. lentus

Alkaline protease S3T / V4I / G61A / V199I / L211 D according to the in SEQ ID NO. 6 indicated amino acid sequence, or that in SEQ ID NO. 5 indicated nucleotide sequence.

Because this showed in tests, as described in the application examples of the present application, the greatest improvement in performance compared to the comparison molecules. It can be obtained as described above.

A preferred embodiment is a protein derived from one of the proteases described above, in particular by fragmentation or deletion mutagenesis, by insertion mutagenesis, by substitution mutagenesis or by fusion of at least one part with at least one other protein.

Such methods have been established in the prior art. Corresponding molecular biological methods are also dealt with in detail, for example, in the textbook Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989.

These include, for example, variants which have been given additional properties via substitution mutagenesis or via further point mutations, which predestine them with regard to specific possible uses, for example through changes in the surface charges as disclosed in WO 00/36069 A1, or through changes in catalysis or loops involved in substrate bindings, as disclosed for example in WO 99/27082 A1. Larger sections of the variants can also be subjected to mutagenesis. For example, the goal of fragment formation or deletion mutagenesis may be to select specific partial functions of the protease or, on the contrary, to exclude it, for example substrate binding, or the interactions with other compounds that are exerted over certain regions of the molecule.

Proteases according to the invention can be provided with additional functions by insertion, substitution or fusion. This includes, for example, coupling to certain domains, for example binding to cellulose-binding domains, as described, for example, in publications WO 99/57154 A1 to WO 99/57157 A1 is described. The amino acid linkers referred to herein can be realized by forming a single fusion protein from protease, linker region and binding domain. Such a binding domain could also originate from the same or a different protease, for example in order to increase the binding of the protein according to the invention to a protease substrate. This increases the local protease concentration, which can be advantageous in individual applications, for example in the treatment of raw materials.

According to a further embodiment, the protein according to the invention is characterized in that it is additionally derivatized.

This serves in particular to optimize for their respective application. This includes chemical modifications as described, for example, in the application DE 4013142 A1. They can also be modified, for example, by coupling lower or higher molecular weight chemical compounds, such as those naturally carried out by various organisms in connection with protein biosynthesis, such as, for example, the binding of a fatty acid residue near the N-terminus or glycosylations in the synthesis by eukaryotic host cells , Embodiments of the present invention thus represent proteolytic enzymes or fragments which are additionally derivatized.

In connection with the use of proteins according to the invention in washing or cleaning agents, coupling with other washing-active substances or enzymes, for example, is particularly useful. Comparable coupling approaches are described, for example, in patent applications WO 00/18865 A1 and WO 00/57155 A1 for cellulose binding domains. Analogously, couplings to macromolecular compounds, such as polyethylene glycol, can also be carried out in order to modify the molecule with regard to further properties, such as stability or skin tolerance. Such a modification is described, for example, in US Pat. No. 5,230,891 in order to make the proteases in question more suitable for use in cosmetics.

Derivatives of proteins according to the invention can also be understood in the broadest sense to mean preparations of these enzymes. Depending on the extraction, processing or preparation of a protein, it can be mixed with various other substances be socialized, for example from the culture of the producing microorganisms. Culture supernatants from protease-producing microorganisms already show proteolytic activity, which shows that crude extracts can already be used, for example for inactivating other proteinogenic activities.

A protein can also have been specifically mixed with certain other substances, for example to increase its storage stability. All preparations of the actual protein according to the invention are therefore also according to the invention. This is also irrespective of whether or not it actually exhibits this enzymatic activity in a particular preparation. Because it may be desirable that it has little or no activity during storage and that it only develops its proteolytic function at the time of use. This can depend, for example, on the state of folding of the protein or on the reversible binding of one or more accompanying substances from the preparation to a protein according to the invention. In particular, the joint preparation of proteases with protease inhibitors is known from the prior art (WO 00/01826 A2). This also includes fusion proteins in which the inhibitors are linked to the respective proteases via linkers, in particular amino acid linkers (WO 00/01831 A2).

The above-mentioned developments, derivatizations and preparations of proteins according to the invention are particularly desirable if they continue to exert proteolytic activity, because this is the prerequisite for their possible uses according to the invention. The proteases obtained by all types of mutagenesis and / or derivatizations preferably have increased proteolytic activity compared to the starting molecule or the non-derivatized molecule and very particularly improved performance with regard to their intended technical field of application. This includes in particular the improvement of their washing and / or cleaning performance for use in washing or cleaning agents.

This is possible, for example, by combining the point mutations according to the invention with further point mutations which relate to the catalyzed reaction, for example in the active center. For example, using the teaching from application WO 95/30011 A2, it would be possible to use proteases according to the invention, which are those derived from Bacillus fe / rtus subtilisin, in the loop Mutate regions or insert additional amino acids. Such works are described in the applications which have been published under the numbers WO 00/37599 A1, WO 00/37621 A1 to WO 00/37627 A1 and WO 00/71683 A1 to WO 00/71691 A1.

The deletion of a region of the enzyme that interacts with other active substances in the reaction medium and thus impairs the overall reaction, for example via folding effects, could represent such a desired further development. Analogously, fusion with other active enzymes, for example with other proteases, is conceivable in order to achieve an increased hydrolysis rate.

For example, the reversible blocking of proteolytic activity during storage by binding an inhibitor can prevent autoproteolysis and thus cause a high rate of proteolysis in the reaction medium at the time of dilution. For example, coupling to specific binding domains during the cleaning process can increase the concentration of the protease near the substrate relative to that in the liquor and thus increase the contribution of the enzyme to the performance of the agent.

In a further embodiment, the proteins or derivative mentioned are characterized in that they are additionally stabilized or have more than one of the stabilizations shown above.

For reasons of practicability, such methods, which are based on point mutagenesis, are particularly relevant to the invention. All of the options already explained above can also be used in combination on variants according to the invention. Because according to WO 89/09819 A1 it can be assumed that several stabilizing mutations have an additive effect. For example, variants according to the invention which are already stabilized by one of the two amino acids 3T or 41, or by both, can additionally be stabilized by coupling to a polymer or in another manner described above.

The second subject of the invention is formed by nucleic acids. These include, in each case correspondingly preferred, the nucleic acids which code for the proteins or derivatives of the first subject matter of the invention. Because nucleic acids form the starting point for almost all common molecular biological studies and further developments as well as the production of proteins. These include in particular the sequencing of the genes and the derivation of the associated amino acid sequence, any type of mutagenesis and the expression of the proteins. Such methods are described, for example, in the Fritsch, Sambrook and Maniatis manual "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989.

At the DNA level, the enzymes essential to the invention can be optimized for various applications using all of the methods generally termed "protein engineering". In particular, the following properties can be achieved at the protein level: an improvement in the oxidation resistance of the derived protein, stability to denaturing agents or proteases, to high temperatures, acidic or strongly alkaline conditions, a change in sensitivity to calcium or other cofactors, a decrease in immunogenicity or the allergenic effect.

Mutated genes according to the invention include, for example, those which are responsible for individual, targeted base exchanges or randomized point mutations, for deletions of individual bases or partial sequences, fusions with other genes or gene fragments or inversions. Such mutations or modifications can predestine the enzyme derived from the nucleic acids in question for specific applications. Such a mutagenesis can be carried out in a targeted manner or by means of random methods, for example with a subsequent detection and / or selection process (screening and selection) aimed at the activity, on the cloned genes.

In particular in the case of the nucleic acids which code for protein fragments, all three reading frames must be taken into account both in sense and in atrf / sense orientation. Such oligonucleotides can be used as starting points for the synthesis of related nucleic acids via the polymerase chain reaction (PCR). Such oligonucleotides, especially if they cover one of the regions which correspond to the five amino acid positions 3, 4, 61 199 and / or 211, are expressly stated in included the scope of the present invention. This also applies to those which have variable sequences precisely in these positions, so that within a population of many primers there can also be at least one who is responsible for such a position for one of the SEQ ID NO. 3 and / or SEQ ID NO. 5 corresponding partial sequence encoded. The same applies to> Aπf / ^' seπse oligonucleotides, which can be used for example for regulation of expression.

The further development of the proteases according to the invention can be seen in particular in the "Protein engineering" publication by P.N.Bryan (2000) in Biochim. Biophys. Acta, Volume 1543, pp. 203-222, are the considerations presented.

The representatives of this subject matter of the invention are preferably nucleic acids coding for subtilisin proteases, the nucleotide sequence of which corresponds to one of the sequences shown in SEQ ID NO. 3 or SEQ ID NO. 5 specified nucleotide sequences matches. This correspondence particularly affects the regions which correspond to the amino acid sequences SEQ ID NO. 4 or SEQ ID NO. Code 6 for isoleucine in position 199, for aspartic acid in position 211, for threonine in position 3 and / or for isoleucine in position 4, and especially for alanine in position 61 or include these areas.

Because, as shown above and in the examples, these positions indicate particularly preferred representatives of alkaline proteases according to the invention. This teaching is suitably transferred to other subtilisins by mutating the other molecules in question in one or more of these positions. This is expediently carried out according to methods known per se (see above) at the nucleic acid level.

This preferably applies to the nucleic acids which can be derived from a sequence for a Bacillus lentus protease, and in particular from a sequence for a Bacillus lentus DSM 5483 protease. In very particularly preferred cases, the nucleic acid codes for one of the variants β according to the invention. / ertfus-Alkaline protease S3TΛ 4I / G61A / V199I or ß. e / i us-Alkaline protease S3TΛ / 4I / G61A / V199I / L211 D and / or matches one of those in SEQ ID NO. 3 or SEQ ID NO. 5 specified nucleotide sequences. This correspondence relates to that, this Variants characterizing areas and particularly preferably on the complete sequences.

The protected area also includes, for example, those nucleic acids which code for proteolytically active insertion or fusion mutants. For example, the areas responsible for this activity can be fused with cellulose-binding domains or carry point mutations in catalytically inactive areas in order to enable the coupling of the derived protein to a polymer or to reduce its allergenicity.

Vectors form their own subject matter of the invention. These include vectors which contain one of the previously mentioned nucleic acid regions and in particular contain a nucleic acid region which codes for one of the above-mentioned proteins or derivatives.

Because in order to deal with the nucleic acids relevant to the invention, they are suitably ligated in vectors. These are described in detail in the prior art and are commercially available in large numbers and in a wide range, both for cloning and for expression. These include, for example, vectors which are derived from bacterial plasmids, from viruses or from bacteriophages, or predominantly synthetic vectors. They form suitable starting points for molecular biological and biochemical investigations, the expression of the gene in question or the associated protein.

The vectors according to the invention are preferably cloning vectors which contain one of the previously mentioned nucleic acid regions and in particular contain a nucleic acid region which code for one of the above-mentioned proteins or derivatives.

In addition to storage, biological amplification or selection of the gene of interest, cloning vectors are suitable for the molecular biological characterization of the gene in question. At the same time, they represent transportable and storable forms of the claimed nucleic acids and are also starting points for molecular biological techniques that are not bound to cells, such as, for example, the PCR or / n-wϊro mutagenesis method. The vectors according to the invention are likewise preferably expression vectors which contain one of the previously mentioned nucleic acid regions and in particular contain a nucleic acid region which codes for one of the above-mentioned proteins or derivatives and enables its biosynthesis.

Such expression vectors are the basis for realizing the corresponding nucleic acids in biological production systems and thus producing the associated proteins. Preferred embodiments of this subject matter of the invention are expression vectors which carry all the genetic elements necessary for expression, for example the natural promoter originally located in front of this gene or a promoter from another organism. These elements can be arranged, for example, in the form of a so-called expression cassette. They are particularly preferably matched to the selected expression system, in particular the host cell (see below).

Cells form a separate subject of the invention, which can be used in any form for the development, modification or production of proteins or derivatives according to the invention. These include in particular cells that contain one of the vectors referred to above or their characteristic regions, either in plasmid or in chromosomal localization.

This is because they enable, for example, the amplification of the corresponding genes, but also their mutagenesis or transcription and translation, and ultimately biotechnological production.

They are preferably host cells which express one of the previously mentioned proteins or derivatives or can be stimulated to express them, in particular using one of the previously mentioned nucleic acid regions, very particularly using an previously mentioned expression vector.

The host cells that form the proteins enable their biotechnological production. For this, they must have received the gene in question, suitably via a vector, that is to say they have been transformed. This vector or its characteristic areas can be present in the host cell extrachromosomally as a separate genetic element or can be integrated into a chromosome.

In principle, all organisms are suitable as host cells, that is to say prokaryotes, eukaryotes or cyanophyta. Preferred are host cells that are genetically easy to handle, for example in terms of transformation with the expression vector and its stable establishment, for example unicellular fungi or bacteria. In addition, preferred host cells are characterized by good microbiological and biotechnological manageability. This applies, for example, to easy cultivation, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. In this way, each protein according to the invention can be obtained from a large number of host organisms. Often the optimal expression systems for the individual case must be determined experimentally from the abundance of different systems available according to the prior art.

Preferred embodiments are those host cells whose activity can be regulated on the basis of corresponding genetic elements, for example by controlled addition of chemical compounds, by changing the cultivation conditions or depending on the respective cell density. This controllable expression enables a very economical production of the proteins of interest. The expression vector and host cell are suitably matched to one another, for example with regard to the genetic elements required for expression (ribosome binding site, promoters, terminators) or codon usage. The latter can be optimized, for example, by replacing those codons in the gene which are only poorly translated by the host in question, with the same meaning in each case, by those which are more common for the respective host.

In a preferred embodiment, the host cell is characterized in that it is a bacterium, in particular one that secretes the protein formed into the surrounding medium.

Because bacteria are characterized by short generation times and low demands on the cultivation conditions. This allows inexpensive procedures to be established become. Bacteria in fermentation technology also have a wealth of experience. For a special production, gram-negative or gram-positive bacteria can be suitable for a variety of reasons, such as nutrient sources, product formation rate, time required, etc., which can be determined experimentally in individual cases.

In Gram-negative bacteria, such as E. coli, a large number of proteins are secreted into the periplasmic space. This can be advantageous for special applications. Gram-positive bacteria, such as Bacilli, on the other hand, release secreted proteins immediately into the nutrient medium surrounding the cells, from which, according to another preferred embodiment, the expressed proteins according to the invention can be purified directly. The application WO 01/81597 even discloses a method by which gram-negative bacteria also remove the expressed proteins.

Bacteria are preferred which are characterized in that they are gram-positive bacteria, in particular that they belong to the genus Bacillus, very particularly the species Bacillus lentus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus alcalophilus.

One embodiment of the present invention uses Bacillus lentus, particularly β. lentus DSM 5483 itself to express (homologously) proteins according to the invention. In contrast, heterologous expression is preferred. For this purpose, bacteria of the Bacillus genus are preferred because they are best characterized in terms of production technology among the gram-positive bacteria. These include in particular those of the species Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis or other species or strains of Bacillus alcalophilus. Because these species related to Bacillus lentus have a similar codon usage and form comparable subtilisins themselves, so they naturally have a correspondingly aligned synthesis apparatus.

A further advantage can be that a mixture of proteins according to the invention with the subtilisins formed endogenously by the host strains can be obtained via this method. Such a co-expression of ß. fenfr / s-alkaline protease in Bacillus licheniformis ATCC 53926, for example, in the application WO 91/02792 (EP 493398 B1); there are also numerous possible expression vectors disclosed. These systems can also be applied to the newly found variants according to the invention.

Further preferred are host cells which are characterized in that they are eukaryotic cells, in particular those which modify the protein formed post-translationally.

Examples of suitable eukaryotes are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. The modifications that such systems carry out particularly in connection with protein synthesis include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce allergenicity.

A separate subject of the invention are processes for the production of a proteolytic enzyme or derivative according to the invention.

Methods are therefore claimed for the production of a proteolytic enzyme or derivative described above using a nucleic acid described above and / or using a vector referred to above and / or using one of the host cells referred to above.

For example, on the basis of the DNA and amino acid sequences described above, as can also be derived from the sequence listing, for example, corresponding oligopeptides and oligonucleotides up to the complete genes and proteins can be synthesized according to known molecular biological methods. Starting from the known subtilisin-producing microorganisms, other natural producers of subtilisins can also be isolated, whose subtilisin sequences are determined and further developed in accordance with the specifications made here. Such bacterial species can also be cultivated and used for corresponding manufacturing processes. Analogously, new expression vectors can be developed along the lines of the vectors disclosed, for example, in application WO 91/02792. Embodiments of the present invention can also be cell-free on the basis of the associated nucleic acid sequences Expression systems in which protein biosynthesis is reproduced in vitro. All of the elements already explained above can also be combined to form new methods for producing proteins according to the invention. A large number of possible combinations of process steps is conceivable for each protein according to the invention, so that optimal processes must be determined experimentally for each specific individual case.

Agents with a proteolytic enzyme according to the invention described above are a separate subject of the invention. In particular, they are detergents or cleaning agents, very particularly in an amount of 2 μg to 20 mg per g of the agent.

Practically all technical possible uses of enzymes according to the invention depend on the fact that the functional enzyme is used in a corresponding medium. For example, the microbiological possible uses require agents in which the enzyme is usually brought together with the necessary reaction partners or cofactors in the form of highly pure preparations. Agents for the treatment of raw materials or cosmetic preparations are also characterized by specific recipes. According to the invention, all of these formulations are to be understood as agents with the enzyme according to the invention.

As a preferred embodiment, detergents or cleaning agents are included in this subject matter of the invention. Because, as shown in the exemplary embodiments of the present application, it was surprisingly found that a subtilisin variant with an exchange in position 61 (numbering according to the B. / enfus alkaline protease) for one of the amino acids alanine, valine, leucine , Isoleucine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, threonine, histidine, lysine or arginine, especially against one of the amino acids alanine, valine, leucine or isoleucine, especially against alanine on various soils of both textiles and hard surfaces results in a significant increase in performance compared to the non-mutated Moelkül. This effect occurs reproducibly both at different temperatures and at different concentrations.

Agents with the variants described above are preferred accordingly. These include in particular those with the ß. fenfr / s alkaline protease variants S3TΛ / 4I / G61AΛ / 199I and S3TΛ 4I / G61A / V199I / L211 D, as well as with the molecules derived from them.

This subject of the invention includes all conceivable types of cleaning agent, both concentrates and agents to be used undiluted; for use on a commercial scale, in the washing machine or for hand washing or cleaning. These include, for example, detergents for textiles, carpets or natural fibers, for which the term detergent is used according to the present invention. These also include, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather; the term cleaning agent is used for such according to the present invention. Any type of cleaning agent represents an embodiment of the present invention, provided that it is enriched with a protein according to the invention.

Embodiments of the present invention include all forms of administration of the agents according to the invention that are established according to the prior art and / or all expedient forms. These include, for example, solid, powder, liquid, gel or pasty agents, possibly also in several phases, compressed or not compressed; this also includes, for example: extrudates, granules, tablets or pouches, both in large containers and packaged in portions.

Agents according to the invention contain enzymes according to the invention in an amount of from 2 μg to 20 mg and increasingly preferably from 5 μg to 17.5 mg, from 20 μg to 15 mg, from 50 μg to 10 mg, from 100 μg to 7.5 mg, from 200 ug to 5 mg and from 500 ug to 1 mg per gram of the composition. This results in from 40 μg to 4 g and increasingly preferably from 50 μg to 3 g, from 100 μg to 2 g, from 200 μg to 1 g and particularly preferably from 400 μg to 400 mg per application.

The protease activity in such agents can be determined by the method described in Tenside, Volume 7 (1970), pp. 125-132. Accordingly, it is given in PE (protease units). The protease activity of the agents can be up to 1,500,000 protease units per gram of preparation. In addition to an enzyme essential to the invention, an agent according to the invention optionally contains further ingredients such as surfactants, e.g. B. nonionic, anionic and / or amphoteric surfactants, and / or bleaching agents, and / or builders, and optionally other conventional ingredients.

The nonionic surfactants used are preferably alkoxylated, advantageously ethoxylated, in particular primary alcohols having preferably 8 to 18 carbon atoms and an average of 1 to 12 moles of ethylene oxide (EO) per mole of alcohol, in which the alcohol radical can be linear or preferably methyl-branched in the 2-position , or can contain linear and methyl-branched radicals in the mixture, as are usually present in oxo alcohol radicals. However, alcohol ethoxylates with linear residues of alcohols of native origin with 12 to 18 carbon atoms, for example from coconut, palm, tallow or oleyl alcohol, and an average of 2 to 8 EO per mole of alcohol are particularly preferred. The preferred ethoxylated alcohols include, for example, C _12-14 alcohols with 3 EO or 4 EO,

with 7 EO, C _13-15 alcohols with 3 EO, 5 EO, 7 EO or 8 EO, C _12-18 alcohols with 3 EO, 5 EO or 7 EO and mixtures of these, such as mixtures of C _12- ι -A! Alcohol with 3 EO and C _12-18 -alcohol with 5 EO. The degrees of ethoxylation given represent statistical averages, which can be an integer or a fraction for a specific product. Preferred alcohol ethoxylates have a narrow homolog distribution (narrow range ethoxylates, NRE). In addition to these nonionic surfactants, fatty alcohols with more than 12 EO can also be used. Examples of this are tallow fatty alcohol with 14 EO, 25 EO, 30 EO or 40 EO.

Another class of preferably used nonionic surfactants, which are used either as the sole nonionic surfactant or in combination with other nonionic surfactants, are alkoxylated, preferably ethoxylated or ethoxylated and propoxylated, fatty acid alkyl esters, preferably with 1 to 4 carbon atoms in the alkyl chain, in particular fatty acid methyl ester.

Another class of nonionic surfactants that can advantageously be used are the alkyl polyglycosides (APG). Alkypolyglycosides that can be used satisfy the general formula RO (G) _z , in which R is a linear or branched, in particular methyl-branched, saturated or unsaturated, aliphatic radical having 8 to 22, preferably 12 to 18, C atoms and G is the symbol is that for one Glycose unit with 5 or 6 carbon atoms, preferably for glucose. The degree of glycosylation z is between 1.0 and 4.0, preferably between 1.0 and 2.0 and in particular between 1.1 and 1.4. Linear alkyl polyglucosides, ie alkyl polyglycosides, in which the polyglycosyl radical is a glucose radical and the alkyl radical is an n-alkyl radical are preferably used.

Nonionic surfactants of the amine oxide type, for example N-coconut alkyl-N, N-dimethylamine oxide and N-tallow alkyl-N, N-dihydroxyethylamine oxide, and the fatty acid alkanolamides can also be suitable. The proportion of these nonionic surfactants is preferably not above that of the ethoxylated fatty alcohols, in particular not more than half of them.

Other suitable surfactants are polyhydroxy fatty acid amides of the formula (II),

R1

I

R-CO-N- [Z] (II)

in which RCO stands for an aliphatic acyl radical with 6 to 22 carbon atoms, R ^ for hydrogen, an alkyl or hydroxyalkyl radical with 1 to 4 carbon atoms and [Z] for a linear or branched polyhydroxyalkyl radical with 3 to 10 carbon atoms and 3 to 10 hydroxyl groups. The polyhydroxy fatty acid amides are known substances which can usually be obtained by reductive amination of a reducing sugar with ammonia, an alkylamine or an alkanolamine and subsequent acylation with a fatty acid, a fatty acid alkyl ester or a fatty acid chloride.

The group of polyhydroxy fatty acid amides also includes compounds of the formula (III)

R ¹ -OR ²

I

R-CO-N- [Z] (III)

in which R represents a linear or branched alkyl or alkenyl radical having 7 to 12 carbon atoms, R ^{1 represents} a linear, branched or cyclic alkyl radical or an aryl radical having 2 to 8 carbon atoms and R ^{2 represents} a linear, branched or cyclic alkyl radical or an aryl radical or an oxyalkyl radical with 1 to 8 Carbon atoms, C _1-4 alkyl or phenyl radicals are preferred and [Z] stands for a linear polyhydroxyalkyl radical whose alkyl chain is substituted with at least two hydroxyl groups, or alkoxylated, preferably ethoxylated or propoxylated derivatives of this radical.

[Z] is preferably obtained by reductive amination of a reducing sugar, for example glucose, fructose, maltose, lactose, galactose, mannose or xylose. The N-alkoxy- or N-aryloxy-substituted compounds can, for example, be converted into the desired polyhydroxy fatty acid amides by reaction with fatty acid methyl esters in the presence of an alkoxide as catalyst.

Anionic surfactants used are, for example, those of the sulfonate and sulfate type. The surfactants of the sulfonate type are preferably C _9-13 -alkylbenzenesulfonates, olefin sulfonates, that is to say mixtures of alkene and hydroxyalkanesulfonates and disulfonates such as are obtained, for example, from C _12-18 monoolefins having a terminal or internal double bond by sulfonating with gaseous Sulfur trioxide and subsequent alkaline or acidic hydrolysis of the sulfonation products is considered. Alkanesulfonates which are derived from C ₁₂ are also suitable. ₁₈ -alkanes can be obtained, for example, by sulfochlorination or sulfoxidation with subsequent hydrolysis or neutralization. The esters of sulfo fatty acids (ester sulfonates), for example the α-sulfonated methyl esters of hydrogenated coconut, palm kernel or tallow fatty acids, are also suitable.

Other suitable anionic surfactants are sulfonated fatty acid glycerol esters. Fatty acid glycerol esters are to be understood as meaning the mono-, di- and triesters and their mixtures as obtained in the production by esterification of a monoglycerol with 1 to 3 moles of fatty acid or in the transesterification of triglycerides with 0.3 to 2 moles of glycerol. Preferred sulfonated fatty acid glycerol esters are the sulfonation products of saturated fatty acids having 6 to 22 carbon atoms, for example caproic acid, caprylic acid, capric acid, myristic acid, lauric acid, palmitic acid, stearic acid or behenic acid.

The alk (en) yl sulfates are the alkali and in particular the sodium salts of the sulfuric acid half esters of C ₁₂ -C ₁₈ fatty alcohols, for example from coconut oil alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol or the C ₀ -C ₂₀ - Oxo alcohols and those half esters of secondary alcohols of this chain length are preferred. Also preferred are alk (en) yl sulfates of the chain length mentioned which contain a synthetic, straight-chain alkyl radical prepared on a petrochemical basis and which have a degradation behavior analogous to that of the adequate compounds based on oleochemical raw materials. The C ₁₂ -C ₁₆ alkyl sulfates and C ₁₂ -C ₁₅ alkyl sulfates as well as C ₁₄ -C ₁₅ alkyl sulfates are preferred from the point of view of washing technology. 2,3-Alkyl sulfates are also suitable anionic surfactants.

Also, the Schwefelsäuremonoester the ethoxylated with 1 to 6 moles of ethylene oxide chain or branched C _-21 alcohols such as 2-methyl-branched _C9-11 alcohols containing on average 3.5 mol ethylene oxide (EO) or C ι _12- ₈ - Fatty alcohols with 1 to 4 EO are suitable. Because of their high foaming behavior, they are used in cleaning agents only in relatively small amounts, for example in amounts up to 5% by weight, usually from 1 to 5% by weight.

Other suitable anionic surfactants are also the salts of alkylsulfosuccinic acid, which are also referred to as sulfosuccinates or as sulfosuccinic acid esters and which are monoesters and / or diesters of sulfosuccinic acid with alcohols, preferably fatty alcohols and especially ethoxylated fatty alcohols. Preferred sulfosuccinates contain C _8-18 fatty alcohol residues or mixtures thereof. Particularly preferred sulfosuccinates contain a fatty alcohol residue which is derived from ethoxylated fatty alcohols, which in themselves are nonionic surfactants (description see above). Again, sulfosuccinates whose fatty alcohol residues are derived from ethoxylated fatty alcohols with a narrow homolog distribution are particularly preferred. It is also possible to use alk (en) ylsuccinic acid with preferably 8 to 18 carbon atoms in the alk (en) yl chain or salts thereof.

Soaps are particularly suitable as further anionic surfactants. Saturated fatty acid soaps are suitable, such as the salts of lauric acid, myristic acid, palmitic acid, stearic acid, hydrogenated erucic acid and behenic acid, and in particular soap mixtures derived from natural fatty acids, for example coconut, palm kernel or tallow fatty acids. The anionic surfactants, including the soaps, can be in the form of their sodium, potassium or ammonium salts and also as soluble salts of organic bases, such as mono-, di- or triethanolamine. The anionic surfactants are preferably in the form of their sodium or potassium salts, in particular in the form of the sodium salts.

The total amount of the surfactants in the cleaning or washing agents according to the invention is preferably from 5% by weight to 50% by weight, in particular from 8% by weight to 30% by weight, based on the finished agent ,

Agents according to the invention can contain bleaching agents. Among the compounds which serve as bleaching agents and supply H ₂ O ₂ in water, sodium percarbonate, sodium perborate tetrahydrate and sodium perborate monohydrate are of particular importance. Further usable bleaching agents are, for example, peroxopyrophosphates, citrate perhydrates and H ₂ O ₂ -producing peracidic salts or peracids, such as persulfates or persulfuric acid. The urea peroxohydrate percarbamide can also be used, which can be described by the formula H ₂ N-CO-NH ₂ Η ₂ O ₂ . In particular, when using the agents for cleaning hard surfaces, for example in automatic dishwashing, they can, if desired, also contain bleaching agents from the group of organic bleaching agents, although their use is in principle also possible for agents for textile washing. Typical organic bleaching agents are the diacyl peroxides, such as dibenzoyl peroxide. Other typical organic bleaching agents are peroxy acids, examples of which include alkyl peroxy acids and aryl peroxy acids. Preferred representatives are the peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesium monoperphthalate, the aliphatic or substituted aliphatic peroxyacids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid, pamidocarboxyacid, phthalimidoproacid, N-nonenylamide operadipic acid and N-nonenylamidopersuccinate, and aliphatic and araliphatic peroxydicarboxylic acids, such as 1, 12-diperoxycarboxylic acid, 1, 9-diperoxyazelaic acid, diperoxysebacic acid, di-peroxybrassyl acid, the diperoxyphthalic acid, n-4-decanoic acid, 2-decanoic acid, 2-decanoic acid Terephthaloyl-di (6-aminopercaproic acid) can be used. The bleaching agent content of the compositions can be 1 to 40% by weight and in particular 10 to 20% by weight, advantageously using perborate monohydrate or percarbonate.

In order to achieve an improved bleaching effect when washing at temperatures of 60 ° C. and below, and in particular during laundry pretreatment, the agents can also contain bleach activators. Bleach activators which can be used are compounds which, under perhydrolysis conditions, give aliphatic peroxocarboxylic acids having preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or optionally substituted perbenzoic acid. Suitable substances are those which carry O- and / or N-acyl groups of the number of carbon atoms mentioned and / or optionally substituted benzoyl groups. Polyacylated alkylenediamines, in particular tetraacetylethylene diamine (TAED), acylated triazine derivatives, in particular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated glycolurils, in particular 1,3,4,6 are preferred -Tetraacetylglycoluril (TAGU), N-acylimides, especially N-nonanoyl-succinimide (NOSI), acylated phenolsulfonates, especially π-nonanoyl- or isononanoyloxybenzenesulfonate (n- or iso-NOBS), acylated hydroxycarboxylic acids (such as triethyl carbonic acids), such as triethyl carboxylic acids, such as triethyl carbonate ), Carboxylic anhydrides, especially phthalic anhydride, isatoic anhydride and / or succinic anhydride, carboxylic acid amides, such as N-methyldiacetamide, glycolide, acylated polyhydric alcohols, especially triacetin, ethylene glycol diacetate, isopropenylacetate, 2,5-diacetoxy-2,5-dihydrofuran and the German dihydrofuran DE 196 16 693 and DE 196 16767 known enol esters as well as acetylated sorbitol and mannitol or their in the European patent application Mixtures described in EP 0 525239 (SORMAN), acylated sugar derivatives, in particular pentaacetylglucose (PAG), pentaacetylfructose, tetraacetylxylose and octaacetyllactose, as well as acetylated, optionally N-alkylated glucamine or gluconolactone, triazole or triazole derivatives and / or particulate / caprol, caprol, or particulate / caprol, preferably caprol acylated lactams, for example N-benzoylcaprolactam and N-acetylcaprolactam, which are known from international patent applications WO 94/27970, WO 94/28102, WO 94/28103, WO 95/00626, WO 95/14759 and WO 95/17498. The hydrophilically substituted acylacetals known from German patent application DE 196 16769 and the acyl lactams described in German patent application DE 196 16 770 and international patent application WO 95/14075 are also preferred used. The combinations of conventional bleach activators known from German patent application DE 4443 177 can also be used. Nitrile derivatives such as cyanopyridines, nitrile quats, for example N-alkylammonium acetonitrile, and / or cyanamide derivatives can also be used. Preferred bleach activators are sodium-4- (octanoyloxy) benzenesulfonate, n-nonanoyl or isononanoyloxybenzenesulfonate (n- or iso-NOBS), Undecenoyl- oxybenzenesulfonate (UDOBS), Natriumdodecanoyloxybenzolsulfonat (DOBS), decanoyl oxybenzoic acid (DOBA, OBC 10) and / or dodecanoyloxybenzenesulfonate (OBS 12), and N-methylmorpholinum acetonitrile (MMA). Bleach activators of this type can be used in the customary quantity range from 0.01 to 20% by weight, preferably in amounts from 0.1 to 15% by weight, in particular 1% by weight to 10% by weight, based on the total composition. be included.

In addition to the conventional bleach activators or in their place, so-called bleach catalysts can also be included. These substances are bleach-enhancing transition metal salts or transition metal complexes such as, for example, Mn, Fe, Co, Ru or Mo salt complexes or carbonyl complexes. Mn, Fe, Co, Ru, Mo, Ti, V and Cu complexes with N-containing tripod ligands as well as Co, Fe, Cu and Ru amine complexes are also suitable as bleaching catalysts. preference is given to using those compounds which are described in DE 197 09 284 A1. According to WO 99/63038, acetonitrile derivatives and, according to WO 99/63041, bleach-activating transition metal complex compounds in combination with amylases are also able to develop a bleach-activating effect.

Agents according to the invention generally contain one or more builders, in particular zeolites, silicates, carbonates, organic cobuilders and - where there are no ecological reasons not to use them - the phosphates. The latter are builders to be used particularly in cleaning agents for automatic dishwashing.

Crystalline, layered sodium silicates of the general formula are to be mentioned here

NaMSi _x O _{2x + 1} -yH ₂ O, where M is sodium or hydrogen, x is a number from 1.6 to 4, preferably 1.9 to 4.0 and y is a number from 0 to 20 and preferred values for x 2, 3 or 4. Such crystalline layered silicates are described, for example, in European patent application EP 0 164 514. Preferred crystalline Layered silicates of the formula given are those in which M is sodium and x is 2 or 3. In particular, both ß- and δ-sodium disilicate

Na ₂ Si ₂ O ₅ -yH ₂ O is preferred. Such compounds are commercially available, for example, under the name ^SKS® (from Clariant). This is how SKS-

6 ^® mainly around a δ-sodium disilicate with the formula Na ₂ Si ₂ O ₅ -yH ₂ O, with SKS-7 ^® mainly around the β-sodium disilicate. Reaction with acids (for example citric acid or carbonic acid) results in kanemite from the δ-sodium disilicate

NaHSi ₂ O ₅ yH ₂ O, commercially available under the names SKS-9 ^® and SKS-10 ^® (from Clariant). It can also be advantageous to use chemical modifications of these layered silicates. For example, the alkalinity of the layered silicates can be suitably influenced. Layered silicates doped with phosphate or carbonate have changed crystal morphologies compared to δ-sodium disilicate, dissolve faster and show an increased calcium binding capacity compared to δ-sodium disilicate. Layer silicates are of the general empirical formula x Na ₂ O «y SiO ₂ • z P ₂ O ₅ , in which the ratio x to y is a number from 0.35 to 0.6, the ratio x to z is a number from 1.75 to 1200 and the ratio y to z correspond to a number from 4 to 2800, described in patent application DE 19601 063. The solubility of the layer silicates can also be increased by using particularly finely divided layer silicates. Compounds made from crystalline layered silicates with other ingredients can also be used. Compounds with cellulose derivatives, which have advantages in disintegrating action and are used in particular in detergent tablets, and compounds with polycarboxylates, for example citric acid, or polymeric polycarboxylates, for example copolymers of acrylic acid, are to be mentioned in particular.

Amorphous sodium silicates with a modulus Na ₂ O: SiO ₂ of 1: 2 to 1: 3.3, preferably from 1: 2 to 1: 2.8 and in particular from 1: 2 to 1: 2.6, can also be used are delayed in dissolving and have secondary washing properties. The delay in dissolution compared to conventional amorphous sodium silicates can be caused in various ways, for example by surface treatment, compounding, compacting / compression or by overdrying. In the context of this invention, the term “amorphous” is also understood to mean “X-ray amorphous”. This means that the silicates are not sharp in X-ray diffraction experiments X-ray reflections, as are typical for crystalline substances, provide at most one or more maxima of the scattered X-rays, which have a width of several degree units of the diffraction angle. However, it can very well lead to particularly good builder properties if the silicate particles deliver washed-out or even sharp diffraction maxima in electron diffraction experiments. This is to be interpreted as meaning that the products have microcrystalline areas of size 10 to a few hundred nm, values up to max. 50 nm and in particular up to max. 20 nm are preferred. Compacted / compacted amorphous silicates, compounded amorphous silicates and over-dried X-ray amorphous silicates are particularly preferred.

An optionally usable, finely crystalline, synthetic and bound water-containing zeolite is preferably zeolite A and / or P. As zeolite P, zeolite ^MAP® (commercial product from Crosfield) is particularly preferred. However, zeolite X and mixtures of A, X and / or P are also suitable. Commercially available and can preferably be used in the context of the present invention, for example a co-crystallizate of zeolite X and zeolite A (about 80% by weight of zeolite X) ), which is sold by CONDEA Augusta SpA under the brand name VEGOBOND AX ^® and by the formula

nNa ₂ O ^• (1-n) K ₂ O ^• AI ₂ O ₃ ^■ (2 - 2.5) SiO ₂ ^■ (3.5 - 5.5) H ₂ O

can be described. Suitable zeolites have an average particle size of less than 10 μm (volume distribution; measurement method: Coulter Counter) and preferably contain 18 to 22% by weight, in particular 20 to 22% by weight, of bound water.

Of course, it is also possible to use the generally known phosphates as builders, provided that such use should not be avoided for ecological reasons. Of the large number of commercially available phosphates, the alkali metal phosphates, with particular preference for pentasodium or pentapotassium triphosphate (sodium or potassium tripolyphosphate), have the greatest importance in the detergent and cleaning agent industry. Alkali metal phosphates is the general term for the alkali metal (especially sodium and potassium) salts of the various phosphoric acids, in which one can distinguish between metaphosphoric acids (HPO ₃ ) _n and orthophosphoric acid H PO _{4 in} addition to higher molecular weight representatives. The phosphates combine several advantages: they act as alkali carriers, prevent limescale deposits on machine parts or lime incrustations in tissues and also contribute to cleaning performance.

Sodium dihydrogen phosphate, NaH ₂ PO, exists as a dihydrate (density 1.91, preferably ^"3 , melting point 60 °) and as a monohydrate (density 2.04, preferably ^{" 3} ). Both salts are white, water-soluble powders, which lose water of crystallization when heated and at 200 ° C into the weakly acidic diphosphate (disodium hydrogen diphosphate, Na ₂ H ₂ P ₂ O ₇ ), at higher temperature in sodium trimetaphosphate (Na ₃ P ₃ O ₉ ) and Maddrell's salt (see below). NaH ₂ PO _{4 is} acidic; it occurs when phosphoric acid is adjusted to a pH of 4.5 with sodium hydroxide solution and the mash is sprayed. Potassium dihydrogen phosphate (primary or monobasic potassium phosphate, potassium biphosphate, KDP), KH ₂ PO ₄ , is a white salt with a density of 2.33 ^"3 , has a melting point of 253 ° C [decomposition to form potassium polyphosphate (KPO ₃ ) _x ] and is easily soluble in water.

Disodium hydrogen phosphate (secondary sodium phosphate), Na ₂ HPO, is a colorless, very easily water-soluble crystalline salt. It exists anhydrous and with 2 mol. (Density 2.066 gladly ^"3 , water loss at 95 °), 7 mol. (Density 1, 68 gladly ^'3 , melting point 48 ° C with loss of 5 H ₂ O) and 12 mol. Water (Density 1, 52 like ^"3 , melting point 35 ° C with loss of 5 H ₂ O), becomes anhydrous at 100 ° C and changes to diphosphate Na ₄ P ₂ O ₇ when heated more. Disodium hydrogen phosphate is prepared by neutralizing phosphoric acid with soda solution using phenolphthalein as an indicator. Dipotassium hydrogen phosphate (secondary or dibasic potassium phosphate), K ₂ HPO ₄ , is an amorphous, white salt that is easily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na ₃ PO ₄ , are colorless crystals, which like dodecahydrate have a density of 1.62 ^"3 and a melting point of 73-76 ° C (decomposition), as decahydrate (corresponding to 19-20% P ₂ O ₅ ) have a melting point of 100 ° C. and, in anhydrous form (corresponding to 39-40% P ₂ O ₅ ), a density of 2.536 ^{″ 3} . Trisodium phosphate is light in alkaline reaction water soluble and is prepared by evaporating a solution of exactly 1 mol of disodium phosphate and 1 mol of NaOH. Tripotassium phosphate (tertiary or triphase potassium phosphate), K ₃ PO ₄ , is a white, deliquescent, granular powder with a density of 2.56 ^"3 , has a melting point of 1340 ° and is readily soluble in water with an alkaline reaction. It is formed, for example when heating Thomas slag with coal and potassium sulfate Despite the higher price, the more soluble, therefore highly effective, potassium phosphates are often preferred over corresponding sodium compounds in the cleaning agent industry.

Tetrasodium diphosphate (sodium pyrophosphate), Na ₄ P ₂ O ₇ , exists in anhydrous form (density 2.534 like ^"3 , melting point 988 ° C, also given 880 ° C) and as decahydrate (density 1, 815-1, 836 like ^{" 3} , Melting point 94 ° C with loss of water). Both substances are colorless crystals that are soluble in water with an alkaline reaction. Na ₄ P ₂ O ₇ is formed by heating disodium phosphate to> 200 ° C or by reacting phosphoric acid with soda in a stoichiometric ratio and dewatering the solution by spraying. The decahydrate complexes heavy metal salts and hardness formers and therefore reduces the hardness of the water. Potassium diphosphate (potassium pyrophosphate), K ₄ P ₂ O ₇ , exists in the form of the trihydrate and is a colorless, hygroscopic powder with a density of 2.33 ^"3 , which is soluble in water, the pH value being 1% Solution at 25 ° C is 10.4.

Condensation of the NaH ₂ PO or the KH ₂ PO ₄ produces higher molecular weight sodium and potassium phosphates, in which one can distinguish cyclic representatives, the sodium or potassium metaphosphates and chain-like types, the sodium or potassium polyphosphates. A large number of terms are used in particular for the latter: melt or glow phosphates, Graham's salt, Kurrol's and Maddrell's salt. All higher sodium and potassium phosphates are collectively referred to as condensed phosphates.

The technically important pentasodium triphosphate, Na ₅ P ₃ O ₁₀ (sodium tripolyphosphate), is an anhydrous or non-hygroscopic, water-soluble salt of the general formula NaO- [P (O) (ONa) -O] _n that crystallizes with 6 H ₂ O. -Na with n = 3. About 17 g of the salt free of water of crystallization dissolve in 100 g of water at room temperature, about 20 g at 60 ° C and about 32 g at 100 ° C; After heating the solution at 100 ° C. for two hours, hydrolysis produces about 8% orthophosphate and 15% diphosphate. In the Production of pentasodium triphosphate, phosphoric acid is reacted with sodium carbonate solution or sodium hydroxide solution in a stoichiometric ratio and the solution is dewatered by spraying. Similar to Graham's salt and sodium diphosphate, pentasodium triphosphate dissolves many insoluble metal compounds (including lime soaps, etc.). Pentapotassium triphosphate, K ₅ P ₃ O ₁₀ (potassium tripolyphosphate), is commercially available, for example, in the form of a 50% strength by weight solution (> 23% P ₂ O ₅ , 25% K ₂ O). The potassium polyphosphates are widely used in the detergent and cleaning agent industry. There are also sodium potassium tripolyphosphates which can also be used in the context of the present invention. These occur, for example, when hydrolysing sodium trimetaphosphate with KOH:

(NaPO ₃ ) ₃ + 2 KOH -> Na ₃ K ₂ P ₃ O ₁₀ + H ₂ O

According to the invention, these can be used just like sodium tripolyphosphate, potassium tripolyphosphate or mixtures of these two; Mixtures of sodium tripolyphosphate and sodium potassium tripolyphosphate or mixtures of potassium tripolyphosphate and sodium potassium tripolyphosphate or mixtures of sodium tripolyphosphate and potassium tripolyphosphate and sodium potassium tripolyphosphate can also be used according to the invention.

Organic cobuilders which can be used in the washing and cleaning agents according to the invention are, in particular, polycarboxylates or polycarboxylic acids, polymeric polycarboxylates, polyaspartic acid, polyacetals, optionally oxidized dextrins, further organic cobuilders (see below) and phosphonates. These classes of substances are described below.

Usable organic builders are, for example, the polycarboxylic acids which can be used in the form of their sodium salts, polycarboxylic acids being understood to mean those carboxylic acids which carry more than one acid function. For example, these are citric acid, adipic acid, succinic acid, glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid, sugar acids, aminocarboxylic acids, nitrilotriacetic acid (NTA), as long as such use cannot be avoided for ecological reasons, and mixtures of these. Preferred salts are the salts of polycarboxylic acids such as citric acid, adipic acid, succinic acid, glutaric acid, tartaric acid, sugar acids and mixtures of these. The acids themselves can also be used. In addition to their builder effect, they typically also have the property of an acidifying component and thus also serve to set a lower and milder pH of detergents or cleaning agents, unless the pH resulting from the mixture of the other components is desired. In particular, system-friendly and environmentally compatible acids such as citric acid, acetic acid, tartaric acid, malic acid, lactic acid, glycolic acid, succinic acid, glutaric acid, adipic acid, gluconic acid and any mixtures thereof can be mentioned. Mineral acids, in particular sulfuric acid or bases, in particular ammonium or alkali metal hydroxides, can also serve as pH regulators. Such regulators are contained in the inventive compositions in amounts of preferably not more than 20% by weight, in particular from 1.2% by weight to 17% by weight.

Polymeric polycarboxylates are also suitable as builders, for example the alkali metal salts of polyacrylic acid or polymethacrylic acid, for example those with a relative molecular weight of 500 to 70,000 g / mol.

For the purposes of this document, the molecular weights given for polymeric polycarboxylates are weight-average molecular weights M _{w of} the particular acid form, which were determined in principle by means of gel permeation chromatography (GPC), using a UV detector. The measurement was made against an external polyacrylic acid standard, which provides realistic molecular weight values due to its structural relationship to the polymers investigated. This information differs significantly from the molecular weight information for which polystyrene sulfonic acids are used as standard. The molecular weights measured against polystyrene sulfonic acids are generally significantly higher than the molecular weights given in this document.

Suitable polymers are, in particular, polyacrylates, which preferably have a molecular weight of 2,000 to 20,000 g / mol. Because of their superior solubility, the short-chain polyacrylates with molecular weights of 2,000 to 10,000 g / mol, and particularly preferably 3,000 to 5,000 g / mol, can in turn be preferred from this group. Also suitable are copolymeric polycarboxylates, in particular those of acrylic acid with methacrylic acid and of acrylic acid or methacrylic acid with maleic acid. Copolymers of acrylic acid with maleic acid which contain 50 to 90% by weight of acrylic acid and 50 to 10% by weight of maleic acid have proven to be particularly suitable. Their relative molecular weight, based on free acids, is generally 2,000 to 70,000 g / mol, preferably 20,000 to 50,000 g / mol and in particular 30,000 to 40,000 g / mol. The (co) polymeric polycarboxylates can be used either as a powder or as an aqueous solution. The content of (co) polymeric polycarboxylates in the agents can be from 0.5 to 20% by weight, in particular 1 to 10% by weight.

To improve water solubility, the polymers can also contain allylsulfonic acids, such as, for example, allyloxybenzenesulfonic acid and methallylsulfonic acid, as monomers.

Also particularly preferred are biodegradable polymers composed of more than two different monomer units, for example those which contain salts of acrylic acid and maleic acid as well as vinyl alcohol or vinyl alcohol derivatives as monomers or those which contain salts of acrylic acid and 2-alkylallylsulfonic acid and sugar derivatives as monomers.

Further preferred copolymers are those which preferably have acrolein and acrylic acid / acrylic acid salts or acrolein and vinyl acetate as monomers.

Also to be mentioned as further preferred builder substances are polymeric aminodicarboxylic acids, their salts or their precursor substances. Polyaspartic acids or their salts and derivatives are particularly preferred.

Other suitable builder substances are polyacetals, which can be obtained by reacting dialdehydes with polyolcarboxylic acids which have 5 to 7 carbon atoms and at least 3 hydroxyl groups. Preferred polyacetals are obtained from dialdehydes such as glyoxal, glutaraldehyde, terephthalaldehyde and their mixtures and from polyol carboxylic acids such as gluconic acid and / or glucoheptonic acid. Other suitable organic builder substances are dextrins, for example oligomers or polymers of carbohydrates, which can be obtained by partial hydrolysis of starches. The hydrolysis can be carried out by customary, for example acid or enzyme-catalyzed, processes. They are preferably hydrolysis products with average molar masses in the range from 400 to 500,000 g / mol. A polysaccharide with a dextrose equivalent (DE) in the range from 0.5 to 40, in particular from 2 to 30, is preferred, DE being a customary measure of the reducing effect of a polysaccharide compared to dextrose, which has a DE of 100 has. Both maltodextrins with a DE between 3 and 20 and dry glucose syrups with a DE between 20 and 37 as well as so-called yellow dextrins and white dextrins with higher molar masses in the range from 2,000 to 30,000 g / mol can be used.

The oxidized derivatives of such dextrins are their reaction products with oxidizing agents which are capable of oxidizing at least one alcohol function of the saccharide ring to the carboxylic acid function. Particularly preferred organic builders for agents according to the invention are oxidized starches, or their derivatives from the applications EP 472 042, WO 97/25399, and EP 755 944.

Oxydisuccinates and other derivatives of disuccinates, preferably ethylenediamine disuccinate, are further suitable cobuilders. Ethylene diamine N, N'-disuccinate (EDDS) is preferably used in the form of its sodium or magnesium salts. Glycerol disuccinates and glycerol trisuccinates are also preferred in this connection. Suitable amounts used in formulations containing zeolite, carbonate and / or silicate are between 3 and 15% by weight.

Other useful organic cobuilders are, for example, acetylated hydroxycarboxylic acids or their salts, which may also be in lactone form and which contain at least 4 carbon atoms and at least one hydroxyl group and a maximum of two acid groups.

Another class of substances with cobuilder properties are the phosphonates. These are, in particular, hydroxyalkane or aminoalkane phosphonates. Among the hydroxyalkane phosphonates, the 1- Hydroxyethane-1, 1-diphosphonate (HEDP) of particular importance as a cobuilder. It is preferably used as the sodium salt, the disodium salt reacting neutrally and the tetrasodium salt in an alkaline manner (pH 9). Preferred aminoalkane phosphonates are ethylenediaminetetramethylenephosphonate (EDTMP), diethylenetriaminepentamethylenephosphonate (DTPMP) and their higher homologs. They are preferably in the form of the neutral sodium salts, e.g. B. as the hexasodium salt of EDTMP or as the hepta and octa sodium salt of DTPMP. HEDP is preferably used as the builder from the class of the phosphonates. The aminoalkanephosphonates also have a pronounced ability to bind heavy metals. Accordingly, it may be preferred, particularly if the agents also contain bleach, to use aminoalkanephosphonates, in particular DTPMP, or to use mixtures of the phosphonates mentioned.

In addition, all compounds which are able to form complexes with alkaline earth metal ions can be used as cobuilders.

Builder substances can optionally be present in the agents according to the invention in amounts of up to 90% by weight. They are preferably contained in amounts of up to 75% by weight. Detergents according to the invention have builder contents of in particular 5% by weight to 50% by weight. In agents according to the invention for cleaning hard surfaces, in particular for machine cleaning of dishes, the builder substance content is in particular 5% by weight to 88% by weight, with such agents preferably not using any water-insoluble builder materials. In a preferred embodiment of agents according to the invention for in particular machine cleaning of dishes, 20% by weight to 40% by weight of water-soluble organic builders, in particular alkali citrate, 5% by weight to 15% by weight of alkali carbonate and 20% by weight to Contain 40 wt .-% alkali disilicate.

Solvents that can be used in the liquid to gel compositions of detergents and cleaning agents come, for example, from the group of mono- or polyhydric alcohols, alkanolamines or glycol ethers, provided that they are miscible with water in the concentration range indicated. The solvents are preferably selected from ethanol, n- or i-propanol, butanols, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether,

Ethylene glycol mono-n-butyl ether, diethylene glycol methyl ether, diethylene glycol ethyl ether, Propylene glycol methyl, ethyl or propyl ether, dipropylene glycol monomethyl or ethyl ether, diisopropylene glycol monomethyl or ethyl ether, methoxy, ethoxy or butoxytriglycol, 1-butoxyethoxy-2-propanol, 3-methyl-3- methoxybutanol, propylene glycol t-butyl ether and mixtures of these solvents.

Solvents can be used in the liquid to gel detergents and cleaning agents according to the invention in amounts between 0.1 and 20% by weight, but preferably below 15% by weight and in particular below 10% by weight.

To adjust the viscosity, one or more thickeners or thickening systems can be added to the composition according to the invention. These high-molecular substances, which are also called swelling agents, usually absorb the liquids and swell in the process, in order to finally change into viscous real or colloidal solutions.

Suitable thickeners are inorganic or polymeric organic compounds. The inorganic thickeners include, for example, polysilicic acids, clay minerals such as montmorillonites, zeolites, silicas and bentonites. The organic thickeners come from the groups of natural polymers, modified natural polymers and fully synthetic polymers. Such polymers originating from nature are, for example, agar agar, carrageenan, tragacanth, acacia, alginates, pectins, polyoses, guar flour, locust bean gum, starch, dextrins, gelatin and casein. Modified natural substances that are used as thickeners mainly come from the group of modified starches and celluloses. Examples include carboxymethyl cellulose and other cellulose ethers, hydroxyethyl and propyl cellulose, and core meal ether. Fully synthetic thickeners are polymers such as polyacrylic and polymethacrylic compounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines, polyamides and polyurethanes.

The thickeners can be present in an amount of up to 5% by weight, preferably from 0.05 to 2% by weight, and particularly preferably from 0.1 to 1.5% by weight, based on the finished composition ,

The washing and cleaning agent according to the invention can optionally be further conventional ingredients such as sequestering agents, electrolytes and other auxiliaries, such as optical brighteners, graying inhibitors, silver corrosion inhibitors, color transfer inhibitors, foam inhibitors, abrasives, dyes and / or fragrances, and also contain microbial active ingredients and / or UV absorbers.

Textile detergents according to the invention can contain, as optical brighteners, derivatives of diaminostilbenedisulfonic acid or its alkali metal salts. Suitable are, for example, salts of 4,4'-bis (2-anilino-4-morpholino-1, 3,5-triazinyl-6-amino) stilbene-2,2'-disulfonic acid or compounds of similar structure which are used instead of morpholino Group carry a diethanolamino group, a methylamino group, an anilino group or a 2-methoxyethylamino group. Brighteners of the substituted diphenylstyryl type can also be present, for example the alkali salts of 4,4'-bis (2-sulfostyryl) diphenyl, 4,4'-bis (4-chloro-3-sulfostyryl) diphenyl, or 4 - (4-chlorostyryl) -4 '- (2-sulfostyryl) diphenyl. Mixtures of the aforementioned optical brighteners can also be used.

Graying inhibitors have the task of keeping the dirt detached from the textile fibers suspended in the liquor. Water-soluble colloids of mostly organic nature are suitable for this, for example starch, glue, gelatin, salts of ether carboxylic acids or ether sulfonic acids of starch or cellulose or salts of acidic sulfuric acid esters of cellulose or starch. Water-soluble polyamides containing acidic groups are also suitable for this purpose. Starch derivatives other than those mentioned above can also be used, for example aldehyde starches. Cellulose ethers such as carboxymethyl cellulose (sodium salt), methyl cellulose, hydroxyalkyl cellulose and mixed ethers such as methyl hydroxyethyl cellulose, methyl hydroxypropyl cellulose, methyl carboxymethyl cellulose and mixtures thereof, for example in amounts of 0.1 to 5% by weight, based on the agent, are preferably used.

In order to provide silver corrosion protection, silver corrosion inhibitors can be used in dishwashing detergents according to the invention. Such are known from the prior art, for example benzotriazoles, iron (III) chloride or CoSO ₄ . As is known, for example, from European patent EP 0 736084 B1, silver corrosion inhibitors which are particularly suitable for use together with enzymes are manganese, titanium, zirconium, hafnium, vanadium, cobalt or cerium salts and / or complexes in which the metals mentioned are in one of the oxidation states II, IM, IV, V or VI. Examples of such compounds are MnSO ₄ , V ₂ O ₅ , V ₂ O ₄ , VO ₂ , TiOSO ₄ , K ₂ TiF ₆ , K ₂ ZrF ₆ , Co (NO ₃ ) ₂ , Co (NO ₃ ) ₃ , and mixtures thereof.

Soil release agents or soil repellents are mostly polymers which, when used in a detergent, impart dirt-repellent properties to the laundry fiber and / or support the dirt-removing ability of the other detergent components. A comparable effect can also be observed when used in cleaning agents for hard surfaces.

Particularly effective and long-known soil release agents are copolyesters with dicarboxylic acid, alkylene glycol and polyalkylene glycol units. Examples of these are copolymers or mixed polymers of polyethylene terephthalate and polyoxyethylene glycol (DT 16 17 141, or DT 22 00911). The German patent application DT 22 53 063 mentions acidic agents which contain, inter alia, a copolymer of a dibasic carboxylic acid and an alkylene or cycloalkylene polyglycol. Polymers made from ethylene terephthalate and polyethylene oxide terephthalate and their use in detergents are described in German documents DE 28 57 292 and DE 33 24 258 and European patent EP 0 253 567. European patent EP 066944 relates to agents which contain a copolyester of ethylene glycol, polyethylene glycol, aromatic dicarboxylic acid and sulfonated aromatic dicarboxylic acid in certain molar ratios. European or European patent EP 0 185 427 discloses methyl or ethyl end-capped polyesters with ethylene and / or propylene terephthalate and polyethylene oxide terephthalate units and detergents which contain such a soil release polymer. European patent EP 0 241 984 relates to a polyester which, in addition to oxyethylene groups and terephthalic acid units, also contains substituted ethylene units and glycerol units. Polyesters are known from European patent EP 0 241 985 which, in addition to oxyethylene groups and terephthalic acid units, contain 1, 2-propylene, 1, 2-butylene and / or 3-methoxy-1, 2-propylene groups and glycerol units and are combined with C until C ₄ alkyl groups are end group-capped. European patent application EP 0 272033 discloses polyesters with poly-propylene terephthalate and polyoxyethylene terephthalate units which are end-capped by C _1-4 -alkyl or acyl radicals. European patent EP 0274 907 describes sulfoethyl end-capped terephthalate-containing soil-release polyesters. According to the European patent application EP 0357 280 discloses sulfonation of unsaturated end groups in soil-release polyesters with terephthalate, alkylene glycol and poly-C ₂ . ₄ - Glycol units manufactured. International patent application WO 95/32232 relates to acidic, aromatic dirt-releasing polyesters. International polymer application WO 97/31085 discloses non-polymeric soil repellent active ingredients for materials made of cotton with several functional units: a first unit, which can be cationic, for example, is capable of adsorption onto the cotton surface by electrostatic interaction, and a second The unit, which is hydrophobic, is responsible for the active substance remaining at the water / cotton interface.

The dye transfer inhibitors that are suitable for use in textile detergents according to the invention include, in particular, polyvinylpyrrolidones, polyvinylimidazoles, polymeric N-oxides such as poly (vinylpyridine-N-oxide) and copolymers of vinylpyrrolidone with vinylimidazole.

When used in machine cleaning processes, it may be advantageous to add foam inhibitors to the agents. Suitable foam inhibitors are, for example, soaps of natural or synthetic origin, which have a high proportion of C ₁₈ -C ₂ fatty acids. Suitable non-surfactant-like foam inhibitors are, for example, organopolysiloxanes and their mixtures with microfine, optionally signed silica, and paraffins, waxes, microcrystalline waxes and their mixtures with signed silica or bistearylethylenediamide. Mixtures of different foam inhibitors are also used with advantages, for example those made of silicone, paraffins or waxes. The foam inhibitors, in particular silicone and / or paraffin-containing foam inhibitors, are preferably bound to a granular, water-soluble or dispersible carrier substance. Mixtures of paraffins and bistearylethylenediamides are particularly preferred.

A cleaning agent according to the invention for hard surfaces can also contain abrasive components, in particular from the group comprising quartz flours, wood flours, plastic flours, chalks and micro-glass balls and mixtures thereof. Abrasives are preferably not contained in the cleaning agents according to the invention in excess of 20% by weight, in particular from 5% by weight to 15% by weight. Dyes and fragrances are added to detergents and cleaning agents in order to improve the aesthetic impression of the products and, in addition to the washing and cleaning performance, to provide the consumer with a visually and sensorially "typical and distinctive" product. Individual fragrance compounds, for example the synthetic products of the ester, ether, aldehyde, ketone, alcohol and hydrocarbon type, can be used as perfume oils or fragrances. Fragrance compounds of the ester type are, for example, benzyl acetate, phenoxyethyl isobutyrate, p-tert.-butylcyclohexyl acetate, linalyl acetate, dimethylbenzylcarbyl acetate, phenylethyl acetate, linalyl benzoate, benzyl formate, ethyl methylphenyl glycinate, allylcyclohexylpropylate propylate propionate. The ethers include, for example, benzyl ethyl ether, the aldehydes, for example, the linear alkanals with 8-18 C atoms, citral, citronellal, citronellyloxyacetaldehyde, cyclamenaldehyde, hydroxycitronellal, lilial and bourgeonal, and the ketones, for example, the ionones, α-isomethylionone and methyl -cedryl ketone, the alcohols anethole, citronellol, eugenol, geraniol, linalool, phenylethyl alcohol and terpineol, the hydrocarbons mainly include the terpenes such as limonene and pinene. However, preference is given to using mixtures of different fragrances which together produce an appealing fragrance. Such perfume oils can also contain natural fragrance mixtures, such as are obtainable from plant sources, for example pine, citrus, jasmine, patchouly, rose or ylang-ylang oil. Also suitable are muscatel, sage oil, chamomile oil, clove oil, lemon balm oil, mint oil, cinnamon leaf oil, linden blossom oil, juniper berry oil, vetiver oil, olibanum oil, galbanum oil and labdanum oil as well as orange blossom oil, neroliol, orange peel oil and sandalwood oil. The colorant content of detergents and cleaning agents is usually less than 0.01% by weight, while fragrances can make up up to 2% by weight of the entire formulation.

The fragrances can be incorporated directly into the detergents and cleaning agents, but it can also be advantageous to apply the fragrances to carriers, which increase the adhesion of the perfume to the items to be cleaned and ensure a long-lasting fragrance, especially of treated textiles, due to the slower release of the fragrance. Cyclodextrins, for example, have proven useful as such carrier materials, and the cyclodextrin-perfume complexes can additionally be coated with further auxiliaries. A further preferred carrier for fragrances is the zeolite X described, which also contains fragrances instead of or in a mixture with surfactants can record. Preference is therefore given to detergents and cleaning agents which contain the zeolite X described and fragrances which are preferably at least partially absorbed on the zeolite.

Preferred dyes, the selection of which is not difficult for the person skilled in the art, have a high storage stability and insensitivity to the other ingredients of the compositions and to light, and no pronounced substantivity towards textile fibers in order not to dye them.

To combat microorganisms, detergents or cleaning agents can contain antimicrobial agents. Depending on the antimicrobial spectrum and mechanism of action, a distinction is made between bacteriostatics and bactericides, fungistatics and fungicides etc. Important substances from these groups are, for example, benzalkonium chlorides, alkylarylsulfonates, halophenols and phenol mercuric acetate. In the context of the teaching according to the invention, the terms antimicrobial activity and antimicrobial active substance have the customary meaning, as used, for example, by KH Wallhäußer in "Practice of Sterilization, Disinfection - Preservation: Germ Identification - Industrial Hygiene" (5th ed. - Stuttgart; New York: Thieme, 1995 Suitable antimicrobial agents are preferably selected from the groups of alcohols, amines, aldehydes, antimicrobial acids or their salts, carboxylic acid esters, acid amides, phenols, phenol derivatives, diphenyls, diphenylalkanes, Urea derivatives, oxygen, nitrogen acetals and formals, benzamidines, isothiazolines, phthalimide derivatives, pyridine derivatives, antimicrobial surface-active compounds, guanidines, antimicrobial amphoteric compounds, quinolines, 1, 2-dibromo-2,4-dicyanobutane, iodo-2- propyl-but yl-carbamate, iodine, iodophores, peroxo compounds, halogen compounds and any mixtures of the above.

The antimicrobial active ingredient can be selected from ethanol, n-propanol, i-propanol, 1, 3-butanediol, phenoxyethanol, 1, 2-propylene glycol, glycerin, undecylenic acid, benzoic acid, salicylic acid, dihydracetic acid, o-phenylphenol, N-methylmorpholine acetonitrile (MMA), 2-benzyl-4-chlorophenol, 2,2'-methylene-bis- (6-bromo-4-chlorophenol), 4,4'-dichloro-2'-hydroxydiphenyl ether (dichlosan), 2.4 , 4'-trichloro-2'-hydroxydiphenyl ether (trichlosan), chlorhexidine, N- (4-chlorophenyl) -N- (3,4-dichlorophenyl) urea, N, N '- (1, 10- decan-diyldi-1-pyridinyl-4-ylidene) bis (1-octanamine) dihydrochloride, N, N'-bis (4-chlorophenyl) -3,12-diimino-2,4,11, 13-tetraaza-tetradecanediimidamide, glucoprotamines, antimicrobial surface-active quaternary compounds, guanidines including the bi- and polyguanidines, such as 1,6-bis- (2-ethylhexyl-biguanido-hexane) - dihydrochloride, 1,6-di- (N ₁ , N ₁ '-phenyldiguanido-N ₅ , N ₅ ') -hexane-tetrahydochloride, 1, 6-di- (Ni.Ni'-phenyl-N _L Nrmethyldiguanido-Ns.Ns -hexane-dihydrochloride, 1, 6- Di- (N ₁ , N ₁ '-chlorophenyldiguanido-N ₅ , N ₅ ') -hexane-dihydrochloride, 1,6-di- (N ₁ , N ₁ '-2,6-dichlorophenyldiguanido-N ₅ , N ₅ ') hexane dihydrochloride, 1,6-di- [N ₁ , N-ι'-beta- (p-methoxyphenyl) diguanido-N ₅ , N ₅ '] -hexane dihydrochloride, 1.δ-di-Ni .N-i'-alpha-methyl-.beta.-phenyldiguanido-N ₅ , N ₅ ') -hexane-dihydrochloride, l ^ -Di ^ N ^ N ^ -p-nitrophenyldiguanido-N ₅ , N ₅ ') hexane -dihydrochloride, omega: omega-di- (Nι, N-ι'- phenyldiguanido-N ₅ , N ₅ ') -di-n-propylether dihydrochloride, o mega: omega'-di- (N ₁ , N ₁ '-p-chlorophenyldiguanido-N ₅ , N ₅ ') -di-n-propylether-tetrahydrochloride, 1, 6-di- (N ₁ , N ₁ '-2 , 4-dichlorophenyldiguanido-N ₅ , N ₅ ') hexane-tetrahydrochloride, 1, 6-di- (N ₁ , N ₁ ' -p-methylphenyldiguanido-N ₅ , N ₅ ') hexane-dihydrochloride, 1, 6-di - (N ₁ , N ₁ '-2,4,5-trichlorophenyldiguanido-N ₅ , N ₅ ') hexane-tetrahydrochloride, 1, 6-di-tN _! , Ni '-alpha- (p-chlorophenyl) ethyldiguanido-N ₅ , N ₅ '] hexane dihydrochloride, omega: omega-Di- (N ₁ , N ₁ '-p-chlorophenyldiguanido-N ₅ , N ₅ ') m -xylene-dihydrochloride, 1, 12-di- (N ₁ , Ni '-p- chlorophenyldiguanido-N ₅ , N ₅ ') dodecane-dihydrochloride, I O-DKN _L Ni'-phenyldiguanido-N ₅ , N ₅ ') -decane-tetrahydrochloride, 1, 12-di- (N ₁ , N ₁ '-phenyldiguanido- N ₅ , N ₅ ') dodecane-tetrahydrochloride, 1, 6-di- (N ₁ , N ₁ '-o-chlorophenyldiguanido- N ₅ , N ₅ ') hexane-dihydrochloride, 1, 6-di- (N ₁ , N ₁ ' -o-chlorophenyldiguanido-N ₅ , N ₅ ') hexane-tetrahydrochloride, ethylene-bis- (1-tolyl biguanide) , Ethylene bis (p-tolyl biguanide), ethylene bis (3,5-dimethylphenyl biguanide), ethylene bis (p-tert-amylphenyl biguanide), ethylene bis (nonylphenyl biguanide), ethylene bis (phenyl biguanide) ), Ethylene bis (N-butylphenyl biguanide), ethylene bis (2,5-diethoxyphenyl biguanide), ethylene bis (2,4-dimethylphenyl biguanide), ethylene bis (o-diphenyl biguanide), ethylene bis (mixed amyl naphthyl biguanide), N-butyl ethylene bis (phen yl biguanide), trimethylene bis (o-tolyl biguanide), N-butyl trimethyl bis (phenyl biguanide) and the corresponding salts such as acetates, gluconates, hydrochlorides, hydrobromides, citrates, bisulfites, fluorides, polymaleates, N-cocoalkyl sarcosinates, phosphites, Hypophosphites, perfluorooctanoates, silicates, sorbates, salicylates, maleates, tartrates, fumarates, ethylenediaminetetraacetates, iminodiacetates, cinnamates, thiocyanates, arginates, pyromellitates, tetracarboxybutyrates, benzoates, glutarates,

Monofluorophosphates, perfluoropropionates and any mixtures thereof. Halogenated xylene and cresol derivatives such as p-chlorometacresol or p-chloro meta-xylene, as well as natural antimicrobial agents of plant origin (for example from spices or herbs), animal and microbial origin. Preferably antimicrobial surface-active quaternary compounds, a natural antimicrobial active ingredient of plant origin and / or a natural antimicrobial active ingredient of animal origin, most preferably at least one natural antimicrobial active ingredient of plant origin from the group comprising caffeine, theobromine and theophylline as well as essential oils such as eugenol, thymol and geraniol, and / or at least one natural antimicrobial active ingredient of animal origin from the group comprising enzymes such as protein from milk, lysozyme and lactoperoxidase, and / or at least one antimicrobial surface-active quaternary compound with an ammonium, sulfonium, phosphonium, iodonium - Or arsonium group, peroxo compounds and chlorine compounds are used. Substances of microbial origin, so-called bacteriocins, can also be used.

The quaternary ammonium compounds (QAV) suitable as antimicrobial active ingredients have the general formula (R ¹ ) (R ² ) (R ³ ) (R ⁴ ) N ⁺ X ^" , in which R ¹ to R ^{4 have the} same or different CC ₂₂ alkyl radicals , C ₇ -C ₂₈ aralkyl radicals or heterocyclic radicals, two or, in the case of an aromatic integration, such as in pyridine, even three radicals together with the nitrogen atom forming the heterocycle, for example a pyridinium or imidazolinium compound, and X ^"represent halide ions, sulfate ions , Hydroxide ions or similar anions. For an optimal antimicrobial effect, at least one of the residues preferably has a chain length of 8 to 18, in particular 12 to 16, carbon atoms.

QACs can be produced by reacting tertiary amines with alkylating agents, such as methyl chloride, benzyl chloride, dimethyl sulfate, dodecyl bromide, but also ethylene oxide. The alkylation of tertiary amines with a long alkyl radical and two methyl groups is particularly easy, and the quaternization of tertiary amines with two long radicals and one methyl group can also be carried out using methyl chloride under mild conditions. Amines which have three long alkyl radicals or hydroxy-substituted alkyl radicals are not very reactive and are preferably quaternized with dimethyl sulfate.

Suitable QAC are, for example, benzalkonium chloride (N-alkyl-N, N-dimethyl-benzylammonium chloride, CAS No. 8001-54-5), benzalkon B (/ r), p-dichlorobenzyldimethyl-C12- alkylammonium chloride, CAS No. 58390-78-6), benzoxonium chloride (benzyl-dodecyl-bis (2-hydroxyethyl) ammonium chloride), cetrimonium bromide (N-hexadecyl-N, N-trimethylammonium bromide, CAS No. 57-09-0), Benzetonium chloride (N, N-dimethyl-N- [2- [2- [p- (1, 1, 3,3-tetramethylbutyl) phenoxy] ethoxy] ethyl] benzylammonium chloride, CAS No. 121-54-0 ), Dialkyldimethylammonium chloride such as di-n-decyl-dimethyl-ammonium chloride (CAS No. 7173-51-5-5), didecyldi-methylammonium bromide (CAS No. 2390-68-3), dioctyl-dimethyl-ammoniumchloric, 1- Cetylpyridinium chloride (CAS No. 123-03-5) and thiazoline iodide (CAS No. 15764-48-1) and their mixtures. Particularly preferred QAV are the benzalkonium chlorides with C ₈ -C ₁₈ -alkyl radicals, in particular C ₁₂ -C ₁₄ -alkyl-benzyl-dimethyl-ammonium chloride.

Benzalkonium halides and / or substituted benzalkonium halides are for example commercially available as Barquat ^® ex Lonza, Marquat® ^® ex Mason, Variquat ^® ex Witco / Sherex and Hyamine ^® ex Lonza and as Bardac ^® ex Lonza. Other commercially available antimicrobial agents are N- (3-chloroallyl) hexaminium chloride such as Dowicide ^® and Dowicil ^® ex Dow, benzethonium chloride such as Hyamine ^® 1622 ex Rohm & Haas, methylbenzethonium chloride such as Hyamine ^® 10X ex Rohm & Haas, cetylpyridinium chloride such as Cepacolchlorid ex Merrell ,

The antimicrobial active ingredients are used in amounts of from 0.0001% by weight to 1% by weight, preferably from 0.001% by weight to 0.8% by weight, particularly preferably from 0.005% by weight to 0.3% by weight .-% and in particular from 0.01 to 0.2 wt .-% used.

The agents can contain UV absorbers (UV absorbers), which are absorbed onto the treated textiles and improve the lightfastness of the fibers and / or the lightfastness of other formulation components. UV absorbers are organic substances (light protection filters) that are able to absorb ultraviolet rays and release the absorbed energy in the form of longer-wave radiation, for example heat.

Compounds which have these desired properties are, for example, the compounds and derivatives of benzophenone which are active by radiationless deactivation and have substituents in the 2- and / or 4-position. Substituted benzotriazoles, phenyl-substituted acrylates (cinnamic acid derivatives, optionally with cyano groups in the 2-position), salicylates, organic Ni complexes are also substituted as well as natural substances such as umbelliferone and the body's own urocanoic acid. The biphenyl and, above all, stilbene derivatives described, for example in EP 0728749 A and are available as Tinosorb FD ^® ^® or Tinosorb FR ex Ciba commercially. The UV-B absorbers are: 3-benzylidene camphor or 3-benzylidene norcampher and its derivatives, for example 3- (4-methylbenzylidene) camphor, as described in EP 0693471 B1; 4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4- (dimethylamino) benzoate, 2-octyl 4- (dimethylamino) benzoate and 4-

(Dimethylamino) benzoesäureamylester; Esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl 4-methoxycinnamate, 2-ethylhexyl 2-cyano-3,3-phenylcinnamate (octocrylene); Esters of salicylic acid, preferably salicylic acid 2-ethylhexyl ester, salicylic acid 4-isopropylbenzyl ester, salicylic acid homomethyl ester; Derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4'-methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone; Esters of

Benzalmalonic acid, preferably di-2-ethylhexyl 4-methoxybenzmalonate; Triazine derivatives, such as, for example, 2,4,6-trianilino- (p-carbo-2'-ethyl-1'-hexyloxy) -1, 3,5-triazine and octyl triazone, as described in EP 0818450 A1 or dioctyl butamido Triazone (Uvasorb® HEB); Propane-1,3-diones such as 1- (4-tert-butylphenyl) -3- (4'methoxyphenyl) propane-1,3-dione; Ketotricyclo (5.2.1.0) decane derivatives, as described in EP 0694521 B1. Also suitable are 2-phenylbenzimidazole-5-sulfonic acid and its alkali, alkaline earth, ammonium, alkylammonium, alkanolammonium and glucammonium salts; Sulfonic acid derivatives of benzophenones, preferably 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts; Sulfonic acid derivatives of 3-benzylidene camphor, such as 4- (2-oxo-3-bornylidene methyl) benzene sulfonic acid and 2-methyl-5- (2-oxo-3-bornylidene) sulfonic acid and their salts.

Derivatives of benzoylmethane, such as 1- (4'-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione, 4-tert-butyl, are particularly suitable as typical UV-A filters -4'-methoxydibenzoylmethane (Parsol 1789), 1-phenyl-3- (4'-isopropylphenyl) propan-1, 3-dione and enamine compounds as described in DE 19712033 A1 (BASF). The UV-A and UV-B filters can of course also be used in mixtures. In addition to the soluble substances mentioned, insoluble light-protection pigments, namely finely dispersed, preferably nanoized metal oxides or salts, are also suitable for this purpose. Examples of suitable metal oxides are in particular zinc oxide and titanium dioxide and in addition oxides of iron, zirconium, silicon, manganese, aluminum and cerium as well as their mixtures. Silicates (talc), barium sulfate or zinc stearate can be used as salts. The oxides and salts are already used in the form of the pigments for skin-care and skin-protecting emulsions and decorative cosmetics. The particles should have an average diameter of less than 100 nm, preferably between 5 and 50 nm and in particular between 15 and 30 nm. They can have a spherical shape, but it is also possible to use particles which have an ellipsoidal shape or shape which differs from the spherical shape in some other way. The pigments can also be surface-treated, that is to say hydrophilized or hydrophobicized. Typical examples are coated titanium dioxide, such as titanium dioxide T 805 (Degussa) or Eusolex® T2000 (Merck; silicones and particularly preferably trialkoxyoctylsilanes or simethicones are preferred as hydrophobic coating agents. Micronized zinc oxide is preferably used. Other suitable UV light protection filters are see the overview by P. Finkel in SÖFW-Journal 122 (1996), p. 543.

The UV absorbers are usually used in amounts of from 0.01% by weight to 5% by weight, preferably from 0.03% by weight to 1% by weight.

In general, washing or cleaning-active enzymes are also included in the ingredients customary for washing and cleaning agents. At the same time, detergents or cleaning agents which are characterized by additional enzymes in addition to a protein according to the invention are preferred embodiments of the present invention. These include, for example, other proteases, but also oxidoreductases, cutinases, esterases and / or hemicellulases, and particularly preferably lipases, Amylases, cellulases and / or β-glucanases.

Enzymes such as proteases, amylases, lipases or cellulases have been used as active components in detergents and cleaning agents for decades. Their respective contribution to the washing or cleaning performance of the agent in question is the ability to break down protein-containing impurities in the case of protease, the degradation of starch-containing impurities in the case of amylase and the fat-splitting activity in the case of lipase. Cellulases are beyond their dirt-removing, that is, primary washing or cleaning performance, in particular because their contribution to the secondary washing performance of a detergent and because of their fiber effect on textiles preferably used in detergents. The respective hydrolysis products are attacked, dissolved, emulsified or suspended by the other detergent or cleaning agent constituents or, due to their higher solubility, washed out with the washing liquor, so that, if possible, there are synergy effects between the enzymes and the other constituents.

Proteases can have an effect comparable to the contribution to the secondary washing performance of an agent by cellulase on natural fibers, in particular on wool or silk. Due to their effect on the surface structure of such fabrics, they can have a smoothing effect on the material and thus counteract felting.

Additional enzymes extend the cleaning performance of the corresponding agents by their specific enzymatic performance. These include, for example, hemicellulases such as, for example, β-glucanases (WO 99/06515 A1 and WO 99/06516 A1), oxidoreductases such as, for example, laccases (WO 00/39306 A2) or pectin-dissolving enzymes (WO 00/42145 A1), which in particular are used in special detergents.

Enzymes obtained from microorganisms, such as bacteria or fungi, are primarily suitable for use in agents according to the invention. They are obtained in a known manner by means of fermentation processes from suitable microorganisms, which are described, for example, in German patent applications DE 1940488 and DE 2121397, US patent specifications US 3623957, US 4264738, European patent application EP 006638 A2 and international patent application WO 91 / 02792 A1 are described.

A protein according to the invention and / or other proteins contained can be protected, particularly during storage, by stabilizers, for example, against denaturation, decay or inactivation, for example through physical influences, oxidation or proteolytic cleavage.

A group of stabilizers are reversible protease inhibitors which dissociate in the wash liquor when the agent is diluted. Benzamidine hydrochloride and leupeptin are established for this purpose. Borax, boric acids, boronic acids or their salts or esters are frequently used, including above all derivatives with aromatic groups, for example ortho-substituted in accordance with WO 95 / 12655A1, meta-substituted in accordance with WO 92/19707 A1 and para-substituted in accordance with the patent US 5972873 Phenylboronic acids, or their salts or esters. The applications WO 98/13460 A1 and EP 583534 B1 disclose peptide aldehydes, that is to say oligopeptides with a reduced C terminus, specifically those of 2-50 monomers, for the reversible inhibition of detergent and cleaning agent proteases. The peptidic reversible protease inhibitors include ovomucoid (WO 93/00418 A1). For example, the application WO 00/01826 A2 discloses specific, reversible peptide inhibitors for the protease subtilisin for use in agents containing proteases, and WO 00/01831 A2 corresponding fusion proteins of protease and inhibitor.

Further enzyme stabilizers are amino alcohols such as mono-, di-, triethanol- and - propanolamine and their mixtures, aliphatic carboxylic acids up to C ₁₂ , as is known for example from the applications EP 378261 B1 and WO 97/05227 A1, such as succinic acid, other dicarboxylic acids or Salts of the acids mentioned. The application DE 19650537 A1 discloses end group-capped fatty acid amide alkoxylates for this purpose. Certain organic acids used as builders, as disclosed in WO 97/18287 A1, can additionally stabilize an enzyme contained.

Lower aliphatic alcohols, but above all polyols, such as, for example, glycerol, ethylene glycol, propylene glycol or sorbitol are further frequently used enzyme stabilizers. Calcium salts are also used, such as, for example, calcium acetate or the calcium formate disclosed for this purpose in the patent EP 028865 B2, and magnesium salts, for example according to EP 378262 B1.

Polyamide oligomers (WO 99/43780 A1) or polymeric compounds such as lignin (WO 97/00932 A1), water-soluble vinyl copolymers (EP 828762 B1) or, as disclosed in EP 702712 B1, cellulose ethers, acrylic polymers and / or polyamides stabilize the enzyme preparation against physical influences or pH fluctuations. Polymers containing polyamine N-oxide (EP 587550 B1 and EP 581751 B1) act simultaneously as enzyme stabilizers and as Dye transfer inhibitors. Other polymeric stabilizers are the linear C ₈ -C ₁₈ polyoxyalkylenes disclosed in WO 97/05227 A1 in addition to other constituents. As in the applications WO 97/43377 A1 and WO 98/45396 A1, alkylpolyglycosides could stabilize the enzymatic components of the agent according to the invention and even increase their performance. Crosslinked N-containing compounds, as disclosed in WO 98/17764 A1, fulfill a double function as soil release agents and as enzyme stabilizers. Hydrophobic, nonionic polymer in a mixture with other stabilizers according to the application WO 97/32958 A1 has a stabilizing effect on a cellulase, so that such or similar components could also be suitable for the enzyme essential to the invention.

Reducing agents and antioxidants, as disclosed inter alia in EP 780466 A1, increase the stability of the enzymes against oxidative decomposition. Sulfur-containing reducing agents are known, for example, from the patents EP 080748 B1 and EP 080223 B1. Other examples are sodium sulfite (EP 533239 B1) and reducing sugars (EP 656058 B1).

Combinations of stabilizers are also often used, for example made of polyols, boric acid and / or borax in the application WO 96/31589 A1, the combination of boric acid or borate, reducing salts and succinic acid or other dicarboxylic acids in the application EP 126505 B1 or the combination of Boric acid or borate with polyols or polyamino compounds and with reducing salts, as disclosed in EP 080223 B1. According to WO 98/13462 A1, the action of peptide-aldehyde stabilizers is increased by the combination with boric acid and / or boric acid derivatives and polyols and, according to WO 98/13459 A1, is further increased by the additional use of calcium ions.

Agents with stabilized enzyme activities are preferred embodiments of the present invention. Particularly preferred are those with enzymes which are stabilized in several of the ways shown.

Since agents according to the invention can be offered in all conceivable forms, enzymes according to the invention or proteins in all formulations expedient for addition to the respective agents represent respective embodiments of the present invention. These include, for example, liquid formulations, solid granules or capsules.

The encapsulated form lends itself to protect the enzymes or other ingredients from other constituents, such as bleaching agents, or to enable controlled release. Depending on the size of these capsules, a distinction is made between milli, micro and nanocapsules, microcapsules for enzymes being particularly preferred. Such capsules are disclosed, for example, in patent applications WO 97/24177 A1 and DE 19918267 A1. One possible encapsulation method is that the proteins, starting from a mixture of the protein solution with a solution or suspension of starch or a starch derivative, are encapsulated in this substance. Such an encapsulation process is described in the application WO 01/38471 A1 with the title “Process for the Production of Microencapsulated Enzymes”.

In the case of solid agents, the proteins can be used, for example, in dried, granulated and / or encapsulated form. They can be added separately, that is as a separate phase, or with other constituents together in the same phase, with or without compacting. If microencapsulated enzymes are to be processed in solid form, the water can be removed from the aqueous solutions resulting from the workup, such as spray drying, centrifuging or by re-solubilization, using methods known from the prior art. The particles obtained in this way usually have a particle size between 50 and 200 μm.

The enzymes, and also the protein according to the invention, can be added to liquid, gel-like or pasty agents according to the invention, starting from a protein recovery and preparation carried out according to the prior art in concentrated aqueous or non-aqueous solution, suspension or emulsion, but also in gel form or encapsulated or as dried Powder. Such washing or cleaning agents according to the invention are generally produced by simply mixing the ingredients, which can be added in bulk or as a solution to an automatic mixer. In addition to the primary washing performance, the proteases contained in detergents can also fulfill the function of activating other enzymatic constituents by proteolytic cleavage or inactivating them after a corresponding exposure time. Comparable regulatory functions are also possible via the enzyme according to the invention. An embodiment of the present invention is furthermore those compositions with capsules made of protease-sensitive material which, for example, are hydrolyzed by proteins according to the invention at an intended point in time and release their content. A comparable effect can also be achieved with other multiphase agents.

A further embodiment are agents for the treatment of textile raw materials or for textile care, which are characterized in that they contain, alone or in addition to other active ingredients, one of the proteolytic enzymes described above, in particular for fibers or textiles with natural constituents and very particularly for those with wool or silk.

Because especially natural fibers, such as wool or silk, are characterized by a characteristic, microscopic surface structure. As explained in the example of wool in the article by R. Breier in Melliand textile reports from 1.4.2000 (p. 263), this can lead to undesirable effects such as matting in the long term. To avoid such effects, the natural raw materials are treated with agents according to the invention which, for example, help to smooth the flaky surface structure based on protein structures and thus counteract felting. A particularly preferred embodiment is represented by such agents for fibers or textiles with natural components and very particularly with wool or silk.

In a preferred embodiment, the agent with a protease according to the invention is designed so that it can be used regularly as a care agent, for example by adding it to the washing process, applying it after washing or applying it independently of the washing. The desired effect is to maintain a smooth surface structure of the textile and / or to prevent and / or reduce damage to the fabric. A separate subject of the invention are processes for the mechanical cleaning of textiles or hard surfaces, which are characterized in that a proteolytic enzyme according to the invention becomes active in at least one of the process steps.

Processes for the mechanical cleaning of textiles are generally distinguished by the fact that various cleaning-active substances are applied to the items to be cleaned and washed off after the contact time in a number of process steps, or the items to be treated are treated in some other way with a cleaning agent or a solution of this agent. The same applies to processes for the mechanical cleaning of all materials other than textiles, which are summarized under the term hard surfaces. Such methods can be enriched with proteins according to the invention in at least one of the method steps, and then represent embodiments of the present invention.

Preferred are methods in which an enzyme according to the invention in an amount of 40 μg to 4 g and increasingly preferably from 50 μg to 3 g, from 100 μg to 2 g from 200 μg to 1 g and particularly preferably from 400 μg to 400 mg per Application is used.

Since the enzyme according to the invention naturally already has a protein-dissolving activity and also develops this in media which otherwise have no cleaning power, such as, for example, in a mere buffer, a single sub-step of such a method for machine cleaning of textiles can consist, if desired, in addition to stabilizing compounds, The enzyme according to the invention is applied as the only cleaning-active component to salts or buffer substances. This represents a particularly preferred embodiment of the present invention.

Preferred embodiments of this subject matter of the invention are processes for treating textile raw materials or for textile care, which are characterized in that a proteolytic enzyme according to the invention becomes active in at least one of the process steps. This can be, for example, processes in which materials are prepared for processing in textiles, for example for anti-felt finishing, or processes, for example, which are more suitable for cleaning Enrich textiles with a nourishing component. Because of the effect of proteases on certain tissues described above, preferred embodiments are textile raw materials or textiles with natural components, in particular with wool or silk.

A separate subject of the invention is the use of a proteolytic enzyme according to the invention for cleaning textiles or hard surfaces. This is because enzymes according to the invention can be used, in particular in accordance with the processes described above, to remove protein-containing impurities from textiles or hard surfaces. Preferred embodiments are the use outside of a mechanical process, for example hand washing or the manual removal of stains from textiles or hard surfaces.

It is preferred to use an enzyme according to the invention in an amount of 40 μg to 4 g and increasingly preferably from 50 μg to 3 g, from 100 μg to 2 g from 200 μg to 1 g and particularly preferably from 400 μg to 400 mg per application.

A preferred embodiment of this subject of the invention is the use of a proteolytic enzyme according to the invention for activating or deactivating ingredients of washing or cleaning agents. Because, as is known, protein components of washing or cleaning agents can be inactivated by the action of a protease. The present invention relates to using this otherwise rather undesirable effect in a targeted manner. It is also possible that another component is only activated by proteolysis, for example if it is a hybrid protein from the actual enzyme and the corresponding inhibitor, as has been disclosed, for example, in the application WO 00/01831 A2. Another example of such a regulation is one in which an active component for protecting or controlling its activity is encapsulated in a material which is attacked by proteolysis. Proteins according to the invention can thus be used for inactivation, activation or release reactions.

In the following, all other technical processes, uses and associated means located outside of the washing and cleaning problem, regardless of their diversity, are combined to form an object of the invention, provided that they are represented by a protein according to the invention are characterized. This compilation is not to be understood as a final list, but rather compiles the most important uses of proteases according to the invention that are currently to be recognized. If it turns out that further technical fields can be further developed through the use of proteases according to the invention, then these are included in the scope of protection of the present invention.

One embodiment of this subject of the invention is the use of a proteolytic enzyme according to the invention for biochemical or molecular biological analysis, in particular in the context of an enzymatic analysis method. According to the invention and according to Römpp, "Lexikon Chemie" (Version 2.0, Stuttgart / New York: Georg Thieme Verlag, 1999) enzymatic analysis is understood to mean any biochemical analysis which uses specific enzymes or substrates in order to determine substrates in their identity or their concentration on the one hand or enzymes in identity or activity on the other hand.Areas of application are all fields of work related to biochemistry.A preferred embodiment of this subject of the invention shows the use for end group determination in the context of a sequence analysis.

Another embodiment of this subject of the invention is the use of a proteolytic enzyme according to the invention for the preparation, purification or synthesis of natural substances or biological valuable substances. For example, in the course of the purification of natural substances or biological valuable substances it may be necessary to free them of protein impurities. These can be, for example, low molecular weight compounds, all cell contents or storage substances or proteins. This can be carried out both on a laboratory scale and on an industrial scale, for example after the biotechnological production of a valuable substance.

The use of a proteolytic enzyme according to the invention for the synthesis of proteins or other low-molecular chemical compounds occurs in reverse of the reaction which they naturally catalyze, for example when protein fragments are to be linked to one another or amino acids are to be bound to a compound which is not predominantly composed of protein. such Possible uses are presented for example in the application EP 380362 A1.

A further embodiment of this subject of the invention is the use of a proteolytic enzyme according to the invention for the treatment of natural raw materials when these are to be freed from protein impurities. This primarily includes raw materials that cannot be obtained microbiologically, e.g. from agriculture.

A preferred embodiment is the use for surface treatment, and very particularly in a process for treating the economically important raw material leather. In the course of the tanning process, in particular in the step of the alkaline switch (Römpp, "Lexikon Chemie", Version 2.0, Stuttgart / New York: Georg Thieme Verlag, 1999) water-soluble proteins are extracted from the skin material using proteolytic enzymes, for which proteases according to the invention are suitable, in particular if alkaline conditions prevail and denaturing agents are present.

Another embodiment of this subject of the invention is the use of a proteolytic enzyme according to the invention for the production or treatment of raw materials or intermediates in textile production. An example of this is the processing of cotton, which has to be freed from the capsule components in a process called sizing; another is the treatment of wool; The processing of raw silk is similar. Enzymatic processes are superior to comparable chemical processes, particularly with regard to their environmental compatibility.

In a preferred embodiment, proteins according to the invention are used to remove protective layers from textiles, in particular intermediate products or materials, or to smooth their surface before they are further processed in a subsequent processing step.

Another embodiment of this subject of the invention is the use of a protein according to the invention for the treatment of textile raw materials or for textile care, in particular for the surface treatment of wool or silk or mixed textiles containing wool or silk. This applies both to the manufacture of such textiles and to care during use, for example in connection with textile cleaning (see above).

Another embodiment of this subject of the invention is the use of a proteolytic enzyme according to the invention for the treatment of photographic films, in particular for the removal of gelatin-containing or similar protective layers. This is because such protective layers, in particular those made of gelatin emulsions containing silver salt, are used to coat films, such as, for example, X-ray films, which have to be detached from the carrier material after exposure. Proteases according to the invention can be used for this, in particular under alkaline or slightly denaturing reaction conditions.

A separate subject of the invention is the use of a proteolytic enzyme according to the invention for the production of food or feed. Thus, proteases have been used for the production of food since ancient times. An example of this is the use of rennet for the ripening process of cheese or other dairy products. Such processes can be enriched with a protein according to the invention or can be carried out entirely by it. Carbohydrate-rich foods or food raw materials for non-nutritional purposes, such as flour or dextrin, can also be treated with appropriate proteases in order to rid them of accompanying proteins. A protease according to the invention is also suitable for such applications, in particular if it is to be carried out under alkaline or slightly denaturing conditions.

The same applies to the production of animal feed. In addition to a complete liberation of proteins, it may also be of interest here to treat the protein-containing starting materials or mixtures of substances with proteases only for a short time in order to make them easier for the pets to digest.

In a further embodiment of this subject of the invention, proteins according to the invention can be used for cosmetic purposes. Cosmetic agents with a proteolytic enzyme according to the invention are thus claimed, cosmetic processes involving a proteolytic enzyme according to the invention Enzyme and the use of a proteolytic enzyme according to the invention for cosmetic purposes, in particular in the context of corresponding processes or in appropriate agents.

Because proteases also play a crucial role in the cell renewal process of human skin (desquamation) (T. Egelrud et a \., Acta Der / 77. Venerol., Volume T \ _ (1991), pp. 471-474). Accordingly, proteases are also used as bioactive components in skin care products in order to support the breakdown of the desmosome structures which are increased in dry skin, for example according to the applications WO 95/07688 A1 or WO 99/18219 A1. The use of subtilisin proteases, in particular the B. / en us alkaline protease variants already described for cosmetic purposes is described, for example, in WO 97/07770 A1. Proteases according to the invention, in particular those whose activity is controlled, for example after mutagenesis or by adding appropriate substances which interact with them, are also suitable as active components in skin or hair cleansing or care products. Particularly preferred are preparations of these enzymes which are stabilized as described above, for example by coupling to macromolecular carriers (see US Pat. No. 5,230,891) and / or derivatized by point mutations at highly allergenic positions, so that they are more tolerable to humans by humans.

Accordingly, the use of such proteolytic enzymes for cosmetic purposes is also included in this subject of the invention, in particular in appropriate agents, such as shampoos, soaps or washing lotions, or in care products which are offered, for example, in the form of creams. Use in a peeling medicine is also included in this claim.

Examples

example 1

Generation of the proteases according to the invention

All molecular biological work steps follow standard methods, such as those specified in the manual by Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989, or in the international patent application WO 92/21760 A1 ,

Construction of the mutagenesis vector

The mutagenesis was based on the protease variant ß. / enfr / s-Alkaline protease M131 performed. This variant is described in WO 92/21760 A1 and the strain forming it according to this application was deposited under the name Bacillus licheniformis ATCC 68614 with the American Type Culture Collection, Rockville, MD, USA. In this strain, the gene on the plasmid pCB56M131 replicating in Bacillus is contained in an expression cassette consisting of the promoter, the ribosome binding site and the ATG start codon and the 22 amino terminal amino acids of the alkaline protease from Bacillus licheniformis ATCC 53926, fused with the pre -Pro protein and the mutated sequence of the Bacillus lentus DSM 5483-Alkaline protease. The variant ß. / enuvs alkaline protease M131 has the following mutations compared to the native sequence: S3T, V4I, A188P, V193M, V199I.

For mutagenesis, the entire expression cassette was cut out using the restriction enzymes Barn HI and Sac I and cloned into the vector pUC18 (Amersham Pharmacia Biotech, Freiburg), also cut with Barn HI and Sac I. The following mutagenesis steps were then carried out with the vector pUC18M131 thus obtained. The vector pUC18M131 is shown in Figure 2. The expression cassette for ß. / enfr / s-Alkaline protease M131 containing DNA fragment is in SEQ ID NO. 1 documented; SEQ ID NO. 2 shows the amino acid sequence derived therefrom. The one in SEQ ID NO. 1 shown Barn HI - Sac I fragment extends from positions 1 to 1771 in the vector pUC18M131 shown in FIG. 2; the remaining vector areas are identical to those of the starting plasmids pUC18. mutagenesis

First the original sequence of the alkaline protease from Bacillus lentus DSM 5483 in positions 188 and 193 was restored. For this purpose, the QuikChange ^® method from Stratagene (La Jolla, CA, USA) was used according to the manufacturer's instructions. According to this system, a mutated plasmid was generated in a polymerase reaction using in each case two complementary primers containing the mutation. After digestion of the starting plasmid using Dpn I, as prescribed for the QuikChange ^® method, the reaction mixture was transformed into E. coli XL-1 blue. The clones obtained in this way and containing the gene of interest in plasmid localization can optionally be easily recognized by means of a restriction site inserted by the mutation. In any case, checking by DNA sequencing using a conventional kit using the chain termination method is possible. The same procedure was also used for all subsequent mutagenesis steps.

For the conversion of the triplet CCA (proline) coding for the amino acid in position 188 into GCC (alanine), the two primers 5 ^' -TCA CAG TAT GGC GCC GGG CTT GAC ATT-3 ^' and 5-AAT GTC AAG CCC GGC GCC ATA CTG TGA-3 ^' used. In addition to the mutation, these contain a Nar I restriction site which does not change the amino acid sequence.

For the conversion of the triplet ATG (methionine) coding for the amino acid in position 193 into ATT (isoleucine), the two primers 5'-GGG CTT GAC ATT GTG GCA CCC GGG GTA AAC-3 ^' and 5 ^' -GTT TAC CCC GGG TGC were used CAC AAT GTC AAG CCC-3 ^' used. In addition to the mutation, these contain an Xma CI restriction site which does not change the amino acid sequence.

A clone containing the double mutant plasmid then provided the template for the subsequent mutation of the triplet in position 61 GGG (glycine) in GCT (alanine). For this purpose, the two complementary primers with the sequences 5 ^' - CAA GAT GGG AAT GCT CAT GGC ACG CAT - 3 ^' and 5 ^' - ATG CGT GCC ATG AGC ATT CCC ATC TTG - 3 ^{' were} used. The gene for variant B. / etrfus-alkaline protease S3TΛ / 4l / G61AΛ / 199l or was already there. Starting from this mutant, the leucine in position 211 was finally mutated into the amino acid aspartate to generate the second, particularly preferred variant. For this the two complementary primers with the sequences 5 ^' - ACG TAT GCC AGC GAC AAC GGT ACA TCG - 3 ^' and 5 ^' - CGA TGT ACC GTT GTC GCT GGC ATA CGT - 3' were used. The clones obtained were then checked by DNA sequencing.

The DNA sequence of the gene coding for the entire protease of the mutant S3T / V4I / G61A / V199I is in the sequence listing under SEQ ID NO. 3 specified. Of these, the sequence listing under SEQ ID NO. Derive 4 given amino acid sequence. The DNA or protein sequences of the mutant S3T / V4I / G61A / V199I / L211 D are in the sequence listing under SEQ ID NO. 5, or SEQ ID NO. 6 described. These variants are ß because of the wild-type enzyme. lentus DSM 5483 different positions as ß. lentus- alkaline protease S3TΛ 4I / G61A / V199I, or as ß. / enfus-alkaline protease S3TΛ / 4I / G61A / V199I / L211 D.

Expression of the mutants and protease preparation

The expression cassette with the mutated sequence was used as the Barn HI-Sac I fragment instead of the one shown in SEQ ID NO. 1 fragment shown cloned back into the vector pCB56M131 and transformed into Bacillus subtilis DB104. The strain Bacillus subtilis DB 104 has the genotype his, nprR2, / ιprE18, aprA3 (Kawamura, F. and Doi, R.H. (1984), J. Bacteriol., Volume 160, pages 442-444). The DNA was transformed into Bacillus according to the variant of the protoplast method originally developed by Chang and Cohen described in WO91 / 02792 (1979; Molec. Gen. Genet, volume 168, pages 111-115).

Protease-positive clones obtained in this way were checked in 500 ml of MLBSP medium (10 g / l casiton; 20 g / l tryptone, 10 g / l yeast extract, all from Becton Dickinson, Cockeysville; 5 g / l NaCl; 27 g / l sodium succinate; 100 mg / l MgSO ₄ * 7 H ₂ O; 75 mg / l CaCI ₂ ^* 2 H ₂ O; 0.5 μM MnCI ₂ ; 0.5 μM FeSO ₄ ; 2% (w / v ) Glucose; 50 mM PIPES buffer (from a 1 M stock solution with pH 7.2); 75 mM KPO ₄ (from a 1 M stock solution with pH 7.0); pH = 7.0, adjusted with KOH - and 10 μg / ml tetracycline) in 2000 ml shaking flasks for 72 h at 37 ° C. and 200 revolutions per minute. The supernatant obtained after centrifuging off the cells was determined after the Protease activity (according to the method described in Tenside, Volume 7 (1970), pp. 125-132) was used for the subsequent experiments.

Example 2

For the following examples, standardized soiled textiles were used, which had been obtained from the Swiss Material Testing and Testing Institute, St. Gallen, Switzerland (EMPA), or the laundry research institute, Krefeld. In Example 2 the following soiling / textiles were used: A (blood / milk / soot on cotton), B (blood / milk / ink on cotton), C (blood / milk / ink on a polyester-cotton blend) and D ( Egg / soot on cotton).

With this test material, various detergent formulations were launderometrically examined for their washing performance. A liquor ratio of 1:12 was set in each case and washed for 30 min at a temperature of 40 ° C. The dosage was 5.88 g of the respective agent per liter of wash liquor. The water hardness was 16 ° German hardness.

A basic detergent formulation of the following composition (all figures in percent by weight) was used as the control detergent: 4% linear alkylbenzenesulfonate (sodium salt), 4% C ₁₂ -C ₁₈ fatty alcohol sulfate (sodium salt), 5.5 % C, ₂ -C ₁₈ fatty alcohol with 7 EO, 1% sodium soap, 11% sodium carbonate, 2.5% amorphous sodium disilicate, 20% sodium perborate tetrahydrate, 5.5% TAED, 25% zeolite A, 4, 5% polycarboxylate, 0.5% phosphonate, 2.5% foam inhibitor granulate, 5% sodium sulfate, balance: water, optical brightener, salts. The following proteases were added to the various test series in such a way that a final concentration of 2,250 PU of proteolytic activity per liter of wash liquor was obtained: fer / ft / s F49 alkaline protease (WO 95/23221, manufactured by Fa. Biozym, Kundl, Austria), Savinase ^® (. Novozymes A / S, Bagsvaerd, Denmark) and ß the protease of the invention. fer / ft / s-alkaline protease S3T / V4I / G61A / V199I / L211 D.

After washing, the whiteness of the washed fabrics was measured compared to that of barium sulfate, which had been normalized to 100%. The measurement was carried out on a Datacolor SF500-2 spectrometer at 460 nm (UV blocking filter 3), 30 mm aperture, without gloss, light type D65, 10 °, d / 8 °. The results obtained are called Percent remission, i.e. as percentages compared to barium sulfate together with the respective initial values in the following table 2. The mean values from 4 measurements are given. They allow an immediate conclusion to be drawn about the contribution of the enzyme contained in the washing performance of the agent used.

Table 2:

It can be seen that the ß. / en us-Alkaline protease S3TΛ / 4I / G61A / V199I / L211 D shows significantly better washing performance on all soiling than the established proteases ß. / etrfus alkaline protease F49 and Savinase ^® .

Example 3

Cotton fabrics with the same stains A, B and C as in Example 2 were examined in the same way as in Example 2. The difference was that in this example the protease β according to the invention was used in the same detergent formulations. / e / 7 «vs-alkaline protease S3T / V4I / G61A / V199I with the variant ß. / eπfus-alkaline protease S3TΛ / 4I / V199I and the also known proteases B. / enfus-alkaline protease F49 and Savinase ^{® was} compared. They were again so concentrated that there was a final concentration of 2,250 PE of proteolytic activity per liter of wash liquor, and the temperature was again 40 ° C. The measurements and the evaluation of the test series were also carried out as in Example 2. The following Table 3 shows the results obtained.

Table 3:

In the comparison of the two variants ß. / enfr / s-Alkaline protease S3T / V4I / G61A / V199I and ß. / e / 7uvs-Alkaline Protease S3T / V4I / V199I it can be seen that the exchange of the amino acid glycine in position 61 for the aliphatic amino acid alanine has led to an improvement in the performance of the enzyme in a detergent formulation against different soils and on different fabrics. It clearly exceeds the established proteases ß. / er / fc / s-Alkaline protease F49 and Savinase ^® .

Example 4

Containers with hard, smooth surfaces were provided with (A) sissy and (B) egg / milk in a standardized manner and washed at 45 ° C using the normal program of a Miele ^® G 676 household dishwasher. 20 g of detergent were used per rinse cycle; the water hardness was 16 ° German hardness.

The following basic recipe was used as detergent (all figures in percent by weight): 55% sodium tripolyphosphate (calculated as anhydrous), 4% amorphous sodium disilicate (calculated as anhydrous), 22% sodium carbonate, 9% sodium perborate, 2% TAED, 2% non-ionic surfactant, remainder: water, dyes, perfume. This basic The recipe for the different experiments was identical in activity with the different proteases ß. / enfus alkaline protease F49, Savinase ^® , or the protease variant β according to the invention. / enfcts-Alkaline protease S3T / V4I / G61A / V199I / L211D added that there was an activity of 10,000 PE per rinse cycle. This corresponded to approx. 0.1 mg protease protein per g of the detergent concentrate.

After rinsing, the removal of the soiling was determined gravimetrically in percent. For this purpose, the difference between the weight of the soiled and subsequently rinsed vessel and the initial weight of the vessel was compared to the weight difference of the non-rinsed vessel to the initial weight. This relation can be seen as a percent deduction. The results obtained are summarized in Table 4 below. The mean values from 9 measurements are given there. They allow an immediate conclusion on the contribution of the enzyme contained in the cleaning performance of the agent used.

Table 4:

These results show that the β. / enfus-Alkaline Protease S3T / V4I / G61A / V199I / L211 D is superior to the other proteases tested in terms of their cleaning performance in automatic dishwashing detergents, but is at least equal; even with a comparatively low level of activity.

Example 5

Vessels with hard, smooth surfaces were given the same stains as in the previous example and additionally with stains D (lasagna) and rinsed in the same way and again at 45 ° C. The difference was that in this example the protease β according to the invention was used in the same cleaning agent formulations. / enfus-Alkaline protease S3TΛ 4I / G61A / V199I with the variant ß. / enft / s-Alkaline protease S3TΛ 4I V199I and the also known proteases ß. / enfc / s-Alkaline protease F49 and Savinase ^{® was} compared. They were again so concentrated that there was a proteolytic activity of 10,000 PE per wash. The measurements were also carried out as described in the previous example. The results are summarized in Table 5.

Table 5:

It can be seen that the variant ß. / entos alkaline protease S3T / V4I / G61A / V199I of variant ß. / en / s-Alkaline protease S3T / N4I / N199I is also superior in cleaning agents in its contribution to cleaning performance compared to various types of soiling. This increase in performance can only be attributed to the change in position 61. Compared to all tested soiling, it performs better than Savinase ^® ; and compared to soiling C and D, there is also better performance than with ß. / enrus-alkaline protease F49.

Example 6

As in Example 4, vessels were provided with the same soiling as standard and rinsed in the same way with the same detergent formulations, again at 45 ° C. The only difference was that 20,000 PE were used on the respective proteases. This corresponded to approx. 0.2 mg protease in detergent concentrate. The measurement results obtained in the same manner as in Example 5 are summarized in Table 6 below.

Table 6:

Even with higher protease activities used, the higher contribution of the protease according to the invention to the overall cleaning performance of the agent in question compared to the proteases ß established for automatic dishwashing agents is evident. lentus alkaline protease F49 and Savinase ^® .

Example 7

In the same way as in the previous example, vessels with the soiling B and D with detergent formulations were examined again, the ß protease according to the invention. / e / ifc / s-Alkaline protease S3T / V4I / G61A / V199I, the variant ß. / enft / s-Alkaline protease S3TΛ 4I / V199I, which contained B. Ientus-A \ ka \\ sc e protease F49 or Savinase ^® . They were so concentrated that there was a proteolytic activity of 20,000 PE per wash. The temperature was 45 ° C. The measurements were also carried out as described in Example 5. The results obtained are shown in Table 7.

Table 7:

It can be seen that the variant ß. / enft / s-alkaline protease S3TΛ / 4I / G61A / V199I of variant B. / enfr / s-alkaline protease S3T / V4I / V199I in detergents, even in higher concentrations, is superior in its contribution to cleaning performance to various types of soiling. This increase in performance can only be attributed to the change in position 61. Compared to all tested soiling, the performance is sometimes significantly better than that of Savinase ^® ; and compared to the soiling D there is also a better performance than the ß. / etϊuvs-alkaline protease F49.

Description of the figures

Figure 1: Alignment of the amino acid sequences of the ß invention. / e / 7fc / s-Alkaline protease variant with the most important known subtilisins, each in the mature, that is to say processed, form: S3TΛ / 4I / G61A / V199I B. Ientus-A \ ka \\ sc e protease according to the invention variant

S3T / V4I / G61a / V199I; S3T / V4I / G61A / V199I / L211D B. / e / rtus alkaline protease variant according to the invention

S3T / V4I / G61 AΛ / 199I / L211 D;

Subtilisin 309 Subtilisin from Bacillus lentus according to WO 89/06279 A1; Subtilisin PB92 Subtilisin from Bacillus nov. spec. 92 according to EP 283075 A2; Subtilisin Carlsberg Subtilisin from Bacillus licheniformis according to E.L. Smith et. al. (1968), J. Biol. Chem., Volume 243, pp. 2184-2191;

Subtilisin BPN 'Subtilisin from Bacillus amyloliquefaciens according to J.A. Wells et al. (1983), Nucleic Acids Research, Volume 1_1, pp. 7911-7925;

Consensus Positions in the majority of the given sequences.

Figure 2: Mutagenesis vector pUC18M131

The one in SEQ ID NO. 1 Barn HI - Sac I fragment shown extends from positions 1 to 1771 therein; the remaining vector areas are identical to those of the starting plasmid pUC18 (from Amersham Pharmacia Biotech, Freiburg). For the sake of clarity, no other singular interfaces have been included.

Claims

claims

1. Alkaline protease of the subtilisin type, characterized in that, according to the counting of the subtilisin from Bacillus lentus in position 61, an exchange for one of the amino acids alanine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, Has tryptophan, threonine, histidine, lysine or arginine, preferably against one of the amino acids alanine, valine, leucine or isoleucine, particularly preferably against alanine.

2. Alkaline protease according to claim 1, characterized in that it has isoleucine in position 199 according to the counting of the subtilisin from Bacillus lentus.

3. Alkaline protease of the subtilisin type, characterized in that according to the counting of the subtilisin from Bacillus lentus in position 199 isoleucine, in position 211 aspartic acid and in position 61 one of the amino acids alanine, valine, leucine, isoleucine, cysteine, methionine, Phenylalanine, tyrosine, tryptophan, threonine, histidine, lysine or arginine, preferably alanine, valine, leucine or isoleucine, particularly preferably alanine.

4. Alkaline protease according to one of claims 1 to 3, characterized in that it has at least one stabilization.

5. Alkaline protease according to claim 4, characterized in that it has threonine in position 3 according to the counting of the subtilisin from Bacillus lentus.

6. Alkaline protease according to claim 4 or 5, characterized in that it has isoleucine in position 4 according to the counting of the subtilisin from Bacillus lentus.

7. Alkaline protease according to any one of claims 1 to 6, characterized in that it has threonine in position 3, isoleucine in position 4, isoleucine in position 61, alanine in position 199 and isoleucine in position 199 according to the counting of the Bacillus lentus subtilisin.

8. Alkaline protease according to one of claims 1 to 6, characterized in that according to the counting of the subtilisin from Bacillus lentus in position 3 threonine, in position 4 isoleucine, in position 61 alanine, in position 199 isoleucine and in position 211 has aspartic acid ,

9. Alkaline protease according to one of claims 1 to 8, characterized in that it is a subtilisin derived from a Bacillus subtilisin, in particular from Bacillus / enfr / s subtilisin.

10. Alkaline protease according to claim 9, characterized in that it is a subtilisin derived from the subtilisin of Bacillus lentus DSM 5483, in particular β. / errfus-alkaline protease S3TΛ 4I / G61A / V199I according to the in SEQ ID NO. 4 given amino acid sequence.

11. Alkaline protease according to claim 9 or 10, characterized in that it is a subtilisin derived from the subtilisin of Bacillus lentus DSM 5483, in particular β. / enft / s-Alkaline protease S3TΛ / 4I / G61A / V199I / L211D according to the in SEQ ID NO. 6 given amino acid sequence.

12. Protein derived from a protease according to one of claims 1 to 11, in particular by fragmentation or deletion mutagenesis, by insertion mutagenesis, by substitution mutagenesis or by fusion of at least one part with at least one other protein.

13. Protein according to any one of claims 1 to 12, characterized in that it is additionally derivatized.

14. Protein or derivative according to one of claims 1 to 13, characterized in that it is additionally stabilized.

15. Nucleic acid coding for one of the proteins or derivatives referred to in claims 1 to 14.

16. A nucleic acid coding for a subtilisin protease, the nucleotide sequence of which corresponds to one of the sequences shown in SEQ ID NO. 3 or SEQ ID NO. 5 indicated nucleotide sequences agrees, especially in the areas which correspond to the amino acid sequences SEQ ID NO. 4 or SEQ ID NO. Code 6 for isoleucine in position 199, for aspartic acid in position 211, for threonine in position 3 and / or for isoleucine in position 4, and especially for alanine in position 61 or include these areas.

17. Vector which contains a nucleic acid region designated in claims 15 or 16 and in particular contains a nucleic acid region which codes for one of the proteins or derivatives designated in claims 1 to 14.

18. Cloning vector which contains a nucleic acid region designated in claims 15 or 16 and in particular contains a nucleic acid region which codes for one of the proteins or derivatives referred to in claims 1 to 14.

19. Expression vector which contains a nucleic acid region designated in claims 15 or 16 and in particular contains a nucleic acid region which codes for one of the proteins or derivatives designated in claims 1 to 14 and enables its biosynthesis.

20. Cell containing a vector according to any one of claims 17 to 19.

21. Host cell which expresses one of the proteins or derivatives referred to in claims 1 to 14 or can be stimulated to express it, in particular using a nucleic acid region designated in one of claims 15 or 16, very particularly using an expression vector according to claim 20.

22. Host cell according to claim 21, characterized in that it is a bacterium, in particular one which secretes the protein formed into the surrounding medium.

23. Bacterium according to claim 22, characterized in that it is a gram-positive bacterium, in particular that it belongs to the genus Bacillus, very particularly the species Bacillus lentus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus alcalophilus.

24. Host cell according to claim 20 or 21, characterized in that it is a eukaryotic cell, in particular one that post-translationally modifies the protein formed.

25. A method for producing a proteolytic enzyme or derivative according to one of claims 1 to 14 using a nucleic acid according to one of claims 15 or 16 and / or using a vector according to one of claims 17 to 19 and / or using a host cell according to one of claims 20 to 24.

26. Agent, characterized in that it contains a proteolytic enzyme according to one of claims 1 to 14, in particular washing or cleaning agents, very particularly in an amount of 2 μg to 20 mg per g of the agent.

27. Composition according to claim 26, characterized in that it additionally contains further enzymes, in particular other proteases, amylases, cellulases, hemicellulases, oxidoreductases and / or lipases.

28. Agent for the treatment of textile raw materials or for textile care, characterized in that it contains, alone or in addition to other active ingredients, a proteolytic enzyme according to one of claims 1 to 14, in particular for fibers or textiles with natural components and very particularly for those with wool or Silk.

29. Process for the mechanical cleaning of textiles or hard surfaces, characterized in that a proteolytic enzyme according to one of claims 1 to 14 is active in at least one of the process steps, preferably in an amount of 40 μg to 4 g, particularly preferably of 400 μg to 400 mg per application.

30. A process for the treatment of textile raw materials or for textile care, characterized in that a proteolytic enzyme according to one of claims 1 to 14 becomes active in at least one of the process steps, in particular for textile raw materials or textiles with natural components, in particular for those with wool or silk.

31. Use of a proteolytic enzyme according to any one of claims 1 to 14 for cleaning textiles or hard surfaces, preferably in an amount of 40 ug to 4 g, particularly preferably from 400 ug to 400 mg per application.

32. Use of a proteolytic enzyme according to one of claims 1 to 14 for activating or deactivating ingredients of detergents or cleaning agents.

33. Use of a proteolytic enzyme according to any one of claims 1 to 14 for biochemical analysis or for the synthesis of low molecular weight compounds or of proteins.

34. Use of a proteolytic enzyme according to one of claims 1 to 14 for the preparation, purification or synthesis of natural substances or biological valuable substances.

35. Use of a proteolytic enzyme according to one of claims 1 to 14 for the treatment of natural raw materials, in particular for surface treatment, very particularly in a process for the treatment of leather.

36. Use of a proteolytic enzyme according to any one of claims 1 to 14 for the extraction or treatment of raw materials or intermediates in textile manufacture, in particular for removing protective layers on fabrics.

37. Use of a proteolytic enzyme according to one of claims 1 to 14 for the treatment of textile raw materials or for textile care, in particular for the treatment of wool or silk or mixed textiles containing wool or silk.

38. Use of a proteolytic enzyme according to any one of claims 1 to 14 for the treatment of photographic films, in particular for the removal of gelatin-containing or similar protective layers.

39. Use of a proteolytic enzyme according to one of claims 1 to 14 for the production of food or feed.

40. Cosmetics with a proteolytic enzyme according to one of claims 1 to 14 or cosmetic processes involving a proteolytic enzyme according to one of claims 1 to 14 or the use of a proteolytic enzyme according to one of claims 1 to 14 for cosmetic purposes, in particular within the scope thereof Procedure or in appropriate means.